NZ717570B2 - Anti-cgrp compositions and use thereof - Google Patents
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- NZ717570B2 NZ717570B2 NZ717570A NZ71757012A NZ717570B2 NZ 717570 B2 NZ717570 B2 NZ 717570B2 NZ 717570 A NZ717570 A NZ 717570A NZ 71757012 A NZ71757012 A NZ 71757012A NZ 717570 B2 NZ717570 B2 NZ 717570B2
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Abstract
Disclosed is an anti-human calcitonin gene related peptide (CGRP) antibody or antibody and its use for manufacture of a medicament to treat a disease or condition associated with CGRP.
Description
ANTI-CGRP COMPOSITIONS AND USE THEREOF
This is a divisional of New Zealand patent application 618637, dated 21 May 2012, which
is the national phase entry in New Zealand of PCT International application
(published as ). This application claims the benefit
of U.S. Provisional Application No. 61/488,660 (Atty. Docket No. 67858.730300) filed
May 20, 2011, entitled "ANTI-CGRP COMPOSITIONS AND USE THEREOF" all of
which is hereby incorporated by reference in its entirety.
The instant application contains a Sequence Listing which has been submitted in ASCII
format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII
copy, created on May 18, 2012, is named 67858o730301.txt and is 203,815 bytes in size.
BACKGROUND OF THE INVENTION
Field of the Invention
This invention pertains to antibodies and fragments thereof (including Fab
fragments) having binding specificity to human Calcitonin Gene Related Peptide
(hereinafter “CGRP”). Also described are methods of screening for diseases and disorders
associated with CGRP, and methods of preventing or treating diseases and disorders
associated with CGRP by administering said antibodies or fragments thereof.
Description of Related Art
Calcitonin Gene Related Peptide (CGRP) is produced as a multifunctional
neuropeptide of 37 amino acids in length. Two forms of CGRP, the CGRP-alpha and
CGRP-beta forms, exist in humans and have similar activities. CGRP-alpha and CGRP-
beta differ by three amino acids in humans, and are derived from different genes. The
CGRP family of peptides includes amylin, adrenomedullin, and calcitonin, although each
has distinct receptors and biological activities. Doods, H., Curr. Op. Invest. Drugs,
2(9):1261-68 (2001).
CGRP is released from numerous tissues such as trigeminal nerves, which when
activated release neuropeptides within the meninges, mediating neurogenic inflammation
that is characterized by vasodilation, vessel leakage, and mast-cell degradation. Durham,
P.L., New Eng. J. Med., 350 (11):1073-75 (2004). The biological effects of CGRP are
mediated via the CGRP receptor (CGRP-R), which consists of a seven-transmembrane
component, in conjunction with receptor-associated membrane protein (RAMP). CGRP-R
further requires the activity of the receptor component protein (RCP), which is essential for
an efficient coupling to adenylate cyclase through G proteins and the production of cAMP.
Doods, H., Curr. Op. Invest. Drugs, 2(9):1261-68 (2001).
Migraines are neurovascular disorder affecting approximately 10% of the adult
population in the U.S., and are typically accompanied by intense headaches.
Approximately 20-30% of migraine sufferers experience aura, comprising focal
neurological phenomena that precede and/or accompany the event. CGRP is believe to
play a prominent role in the development of migraines. For example, plasma
concentrations of CGRP were identified elevated in jugular venous blood during the
headache phase of migraines, to the exclusion of other neuropeptides. Moreover,
according to Arulmozhi et al, the following has been identified in migraine sufferers: (1) a
strong correlation between plasma CGRP concentrations and migraines; (2) the infusion of
CGRP produced a migraine-like headache; (3) baseline CGRP levels were elevated; and (4)
changes in plasma CGRP levels during migraine attacks significantly correlated with
headache intensity. Arulmozhi, D.K., et al., Vas. Pharma., 43: 176-187 (2005).
One effective treatment for migraines is the administration of triptans, which are
a family of tryptamine-based drugs, including sumatriptan and rizatriptan. Members of this
family have an affinity for multiple serotonin receptors, including 5-HT , 5-HT , and 5-
1B 1D
. Members of this family of drugs selectively constrict cerebral vessels, but also cause
HT1F
vasoconstrictive effects on coronary vessels. Durham, P.L., New Eng. J. Med., 350
(11):1073-75 (2004). There is a theoretical risk of coronary spasm in patients with
established heart disease following administration, and cardiac events after taking triptans
may rarely occur. Noted to be contraindicated for patients with coronary vascular disease.
Similarly, pain may often be addressed through the administration of certain
narcotics or non-steroidal anti-inflammatory drugs (NSAIDs). However, the
administration of these treatments may occur at the cost of certain negative consequences.
NSAIDs have the potential to cause kidney failure, intestinal bleeding, and liver
dysfunction. Narcotics have the potential to cause nausea, vomiting, impaired mental
functioning, and addiction. Therefore, it is desirable to identify alternative treatments for
pain in order to avoid certain of these negative consequences.
CGRP is believed to play a role in a multitude of diseases and disorders,
including but not limited to migraines, headaches, and pain.
For example, CGRP reportedly may correlate to or even pay a causal play a role in
overactive bladder. Evidence that CGRP may correlate to overactive bladder condition
includes the fact that CGRP is present in urinary tract, DRG and spinal cord - (Wharton et
al., 1986 Neurosci (3):727) and also that C-fiber afferents are critical for carrying impulses
involved in micturition to spinal cord (Yoshida et al., 2011 J Pharmacol Sci (112):128).
Further, it has been reported that the intravesical administration of Botox suppresses CGRP
and significantly reduces intercontraction interval in acetic acid - induced bladder pain
model (Chuang et al., 2004 J Urol (172):1529; Chuang et al., 2009 J Urol (182):786)
Evidence that CGRP may play a causal role in this condition is a recent
published patent application containing data purportedly suggesting that an anti-CGRP Ab
disclosed therein reduced the number of bladder contractions in a turpentine-oil – induced
overactive bladder model –(Pfizer )).
Due to the perceived involvement of CGRP in these and other disorders, there
remains a need in the art for compositions and methods useful for preventing or treating
diseases and disorders associated with CGRP, while avoiding adverse side effects. There
remains a need in the art for compositions or methods that reduce or inhibit diseases or
disorders associated with CGRP, such as migraines, headaches, overactive bladder, and
pain. It is an object of the present invention to go someway towards meeting this need
and/or to provide the public with a useful choice.
BRIEF SUMMARY OF THE INVENTION
[0010a] In a first embodiment the invention provides an isolated anti-human CGRP
antibody or antibody fragment comprising:
(a) a V chain comprising the complementarity determining region (CDR) 1
sequence of SEQ ID NO: 25, the CDR 2 sequence of SEQ ID NO: 26, and the CDR 3
sequence of SEQ ID NO: 27, except that one or two of the CDR amino acid residues in
chain has been substituted with another amino acid residue; and
said VL
(b) a V chain comprising the CDR 1 sequence of SEQ ID NO: 28, the CDR 2
sequence of SEQ ID NO: 29, and the CDR 3 sequence of SEQ ID NO: 30, except that one
or two of the CDR amino acid residues in said V chain has been substituted with another
amino acid residue.
[0010b] In a second embodiment the invention provides an isolated anti-CGRP
antibody or antibody fragment comprising:
(a) a V chain comprising the CDR 1 sequence of SEQ ID NO: 55, the CDR 2
sequence of SEQ ID NO: 56, and the CDR 3 sequence of SEQ ID NO: 57, except that one
or two of the CDR amino acid residues in said V chain has been substituted with another
amino acid residue; and
(b) a V chain comprising the CDR 1 sequence of SEQ ID NO: 58, the CDR 2
sequence of SEQ ID NO: 59, and the CDR 3 sequence of SEQ ID NO: 60, except that one
or two of the CDR amino acid residues in said V chain has been substituted with another
amino acid residue,
wherein said anti-human CGRP antibody or antibody fragment specifically binds
CGRP.
[0010c] In a third embodiment the invention provides a use of at least one anti-
human CGRP antibody or antibody fragment according to the invention, for the
manufacture of a medicament to treat a disease or condition associated with cells that bind
or express CGRP.
[0010d] In a fourth embodiment the invention provides a use of at least one anti-
human CGRP antibody or antibody fragment according to the invention, for the
manufacture of a medicament to treat a headache condition.
[0010e] In a fifth embodiment the invention provides a use of at least one anti-
human CGRP antibody or antibody fragment according to the invention, for the
manufacture of a medicament to treat a migraine condition.
[0010f] In a sixth embodiment the invention provides a method of making the anti-
human CGRP antibody or antibody fragment of the invention in a polyploid yeast culture
that stably expresses and secretes into the culture medium at least 10-25 mg/liter of said
antibody, comprising:
(i) introducing at least one expression vector containing one or more heterologous
polynucleotides encoding said antibody operably linked to a promoter and a signal
sequence into a haploid yeast cell;
(ii) producing by mating or spheroplast fusion a polyploidal yeast from said first
and/or second haploid yeast cell;
(iii) selecting polyploidal yeast cells that stably express said antibody; and
(iv) producing stable polyploidal yeast cultures from said polyploidal yeast cells
that stably express said antibody into the culture medium.
[0010g] In a seventh embodiment the invention provides an isolated polynucleotide
comprising a polynucleotide sequence encoding an anti-human CGRP antibody or antibody
fragment of the invention.
[0010h] In an eighth embodiment the invention provides a vector or isolated host cell
comprising the polynucleotide sequence of the invention.
[0010i] In a ninth embodiment the invention provides a pharmaceutical composition
comprising at least one anti-human CGRP antibody or antibody fragment according to the
invention and a pharmaceutically acceptable carrier.
[0010j] In a tenth embodiment the invention provides a diagnostic composition
comprising at least one anti-human CGRP antibody or antibody fragment according to the
invention and a pharmaceutically acceptable carrier.
The present disclosure is directed to specific antibodies and fragments thereof
having binding specificity for CGRP, in particular antibodies having desired epitopic
specificity, high affinity or avidity and/or functional properties. Another embodiment of
this disclsoure relates to the antibodies described herein, comprising the sequences of the
V , V and CDR polypeptides described herein, and the polynucleotides encoding them. A
preferred embodiment of the disclosure is directed to chimeric or humanized antibodies and
fragments thereof (including Fab fragments) capable of binding to CGRP and/or inhibiting
the biological activities mediated by the binding of CGRP to the CGRP receptor (“CGRP-
R”).
In another preferred embodiment of the disclosure, full length antibodies and
Fab fragments thereof are contemplated that inhibit the CGRP-alpha-, CGRP-beta-, and rat
CGRP-driven production of cAMP. In a further preferred embodiment of the disclosure,
full length and Fab fragments thereof are contemplated that reduce vasodilation in a
recipient following administration.
In another embodiment of the disclosure, chimeric or humanized antibodies and
fragments thereof (including Fab fragments) capable of binding to CGRP are useful in
methods directed to reducing, treating, or preventing migraines (with or without aura),
cancer or tumors, angiogenesis associated with cancer or tumor growth, angiogenesis
associated with cancer or tumor survival, weight loss, pain, hemiplagic migraines, cluster
headaches, migrainous neuralgia, chronic headaches, tension headaches, general
headaches, hot flushes, chronic paroxysomal hemicrania, secondary headaches due to an
underlying structural problem in the head or neck, cranial neuralgia, sinus headaches (such
as for example associated with sinusitis), and allergy-induced headaches or migraines. The
antibodies and antibody fragments described herein particularly have utility in treating,
preventing, ameliorating, controlling or reducing the risk of one or more of the following
conditions or diseases: overactive bladder and other urinary conditions including bladder
infection, pain; chronic pain; neurogenic inflammation and inflammatory pain; neuropathic
pain; eye pain; tooth pain; post-surgical pain, trauma related pain, burn related pain,
diabetes; non-insulin dependent diabetes mellitus and other inflammatory autoimmune
disorders, vascular disorders; inflammation; arthritis; bronchial hyperreactivity, asthma;
shock; sepsis; opiate withdrawal syndrome; morphine tolerance; hot flashes in men and
women; allergic dermatitis; psoriasis; encephalitis; brain trauma; epilepsy;
neurodegenerative diseases; skin diseases including pruritis, neurogenic cutaneous redness,
skin rosaceousness and erythema; inflammatory bowel disease, irritable bowel syndrome,
cystitis; dysmenorrhea, and other conditions that potentially may be treated or prevented or
the symptoms ameliorated by antagonism of CGRP receptors. Of particular importance is
the acute or prophylactic treatment of headache, including migraine and cluster headache,
and other pain related conditions as well as overactive bladder.
Also described are, chimeric or humanized antibodies and fragments thereof
(including Fab fragments) capable of binding to CGRP are preferably useful in methods
directed to reducing, treating, or preventing gastro-esophageal reflux, and visceral pain
associated with gastro-esophageal reflux, dyspepsia, irritable bowel syndrome,
inflammatory bowel disease, Crohn’s disease, ileitis, ulcerative colitis, renal colic,
dysmenorrhea, cystitis, menstrual period, labor, menopause, prostatitis, or pancreatitis.
In another embodiment of the disclosure these antibodies and humanized
versions may be derived from rabbit immune cells (B lymphocytes) and may be selected
based on their homology (sequence identity) to human germ line sequences. These
antibodies may require minimal or no sequence modifications, thereby facilitating retention
of functional properties after humanization. A further embodiment of the disclosure is
directed to fragments from anti-CGRP antibodies encompassing V , V and CDR
polypeptides, e.g., derived from rabbit immune cells and the polynucleotides encoding the
same, as well as the use of these antibody fragments and the polynucleotides encoding
them in the creation of novel antibodies and polypeptide compositions capable of binding
to CGRP and/or CGRP/CGRP-R complexes.
The disclosure also contemplates conjugates of anti-CGRP antibodies and
binding fragments thereof conjugated to one or more functional or detectable moieties.
The disclosure also contemplates methods of making said chimeric or humanized anti-
CGRP or anti-CGRP/CGRP-R complex antibodies and binding fragments thereof. In one
, Fv, scFv
embodiment, binding fragments include, but are not limited to, Fab, Fab', F(ab')2
fragments, SMIPs (small molecule immunopharmaceuticals), camelbodies, nanobodies,
and IgNAR.
Embodiments of the disclosure pertain to the use of anti-CGRP antibodies and
binding fragments thereof for the diagnosis, assessment and treatment of diseases and
disorders associated with CGRP or aberrant expression thereof. The disclosure also
contemplates the use of fragments of anti-CGRP antibodies for the diagnosis, assessment
and treatment of diseases and disorders associated with CGRP or aberrant expression
thereof. Other embodiments of the disclosure relate to the production of anti-CGRP
antibodies or fragments thereof in recombinant host cells, for example mammalian cells
such as CHO, NSO or HEK 293 cells, or yeast cells (for example diploid yeast such as
diploid Pichia) and other yeast strains.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Figure 1 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab1.
Figure 2 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab2.
Figure 3 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab3.
Figure 4 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab4.
Figure 5 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab5.
Figure 6 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab6.
Figure 7 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab7.
Figure 8 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab8.
Figure 9 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab9.
Figure 10 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab10.
Figure 11 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab11.
Figure 12 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab12.
Figure 13 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab13.
Figure 14 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab14.
Figure 15 provides the CGRP-alpha ELISA binding data obtained following the
protocol in Example 1 infra for antibodies Ab1, Ab2, Ab3, and Ab4.
Figure 16 provides the CGRP-alpha ELISA binding data obtained following the
protocol in Example 1 infra for antibodies Ab5, Ab6, Ab7, and Ab8.
Figure 17 provides the CGRP-alpha ELISA binding data obtained following the
protocol in Example 1 infra for antibodies Ab9, Ab10, and Ab14.
Figure 18 provides the CGRP-alpha ELISA binding data obtained following the
protocol in Example 1 infra for antibodies Ab11, Ab12, and Ab13.
Figure 19 demonstrates the inhibition of CGRP-alpha-driven cAMP production
by antibodies Ab1, Ab2, and Ab4, obtained following the protocol in Example 1 infra.
Figure 20 demonstrates the inhibition of CGRP-alpha-driven cAMP production
by antibody Ab3, obtained following the protocol in Example 1 infra.
Figure 21 demonstrates the inhibition of CGRP-alpha-driven cAMP production
by antibodies Ab5 and Ab6, obtained following the protocol in Example 1 infra.
Figure 22 demonstrates the inhibition of CGRP-alpha-driven cAMP production
by antibodies Ab7, Ab8, Ab9, and Ab10, obtained following the protocol in Example 1
infra.
Figure 23 demonstrates the inhibition of CGRP-alpha-driven cAMP production
by antibodies Ab11, Ab12, and Ab13, obtained following the protocol in Example 1 infra.
Figure 24 demonstrates the inhibition of CGRP-alpha-driven cAMP production
by antibody Ab14, obtained following the protocol in Example 1 infra.
Figure 25 demonstrates the inhibition of CGRP-beta-driven cAMP production
by antibodies Ab1, Ab2, and Ab3, obtained following the protocol in Example 1 infra.
Figure 26 demonstrates the inhibition of CGRP-beta-driven cAMP production
by antibodies Ab4, Ab5, and Ab6, obtained following the protocol in Example 1 infra.
Figure 27 demonstrates the inhibition of CGRP-beta-driven cAMP production
by antibodies Ab7 and Ab8, obtained following the protocol in Example 1 infra.
Figure 28 demonstrates the inhibition of CGRP-beta-driven cAMP production
by antibodies Ab9, Ab10, and Ab14, obtained following the protocol in Example 1 infra.
Figure 29 demonstrates the inhibition of CGRP-beta-driven cAMP production
by antibodies Ab11, Ab12, and Ab13, obtained following the protocol in Example 1 infra.
Figure 30 demonstrates the inhibition of rat CGRP-driven cAMP production by
antibodies Ab1, Ab2, Ab4, and Ab5, obtained following the protocol in Example 1 infra.
Figure 31 demonstrates the inhibition of rat CGRP -driven cAMP production by
antibodies Ab3 and Ab6, obtained following the protocol in Example 1 infra.
Figure 32 demonstrates the inhibition of rat CGRP-driven cAMP production by
antibodies Ab7 and Ab8, obtained following the protocol in Example 1 infra.
Figure 33 demonstrates the inhibition of rat CGRP-driven cAMP production by
antibody Ab9, obtained following the protocol in Example 1 infra.
Figure 34 demonstrates the inhibition of rat CGRP-driven cAMP production by
antibody Ab10, obtained following the protocol in Example 1 infra.
Figure 35 demonstrates the inhibition of rat CGRP-driven cAMP production by
antibodies Ab11 and Ab12, obtained following the protocol in Example 1 infra.
Figure 36 demonstrates the inhibition of rat CGRP-driven cAMP production by
antibody Ab13, obtained following the protocol in Example 1 infra.
Figure 37 demonstrates the inhibition of rat CGRP-driven cAMP production by
antibody Ab14, obtained following the protocol in Example 1 infra.
Figure 38 demonstrates the inhibition of binding of radiolabeled CGRP to
CGRP-R by antibodies Ab1-Ab13, obtained following the protocol in Example 6 infra.
Figure 39 demonstrates a reduction in vasodilation obtained by administering
antibodies Ab3 and Ab6 following capsaicin administration in a rat model, relative to a
control antibody, obtained following the protocol in Example 7 infra.
Figure 40 demonstrates a reduction in vasodilation obtained by administering
antibody Ab6 at differing concentrations following capsaicin administration in a rat model,
relative to a control antibody, obtained following the protocol in Example 7 infra.
A-C shows the beneficial effect of Ab3 on bladder capacity during saline
infusion. Animals were administered Ab3 or a negative control antibody, then monitored
during infusion of saline into the bladder. ICI (panel A) was increased and MF (panel B)
was decreased, indicating increased bladder capacity. Differences in AM (panel C) were
within the standard deviation and not statistically significant. Asterisks indicate
statistically significant improvement (p < 0.05 unpaired Student’s t-test, comparison to
Negative control Ab). Legend: filled bars: Ab3 treated (10 mg/kg); open bars: negative
control antibody (10 mg/kg). Error bars indicate the standard deviation. Abbreviations: ICI
: Intercontraction Interval; MF : Micturition Frequency; AM : Amplitude of Micturition.
shows the effect Ab2 in a model of neuropathic pain. Mechanical
allodynia was induced by Chung surgery (L5/L6 spinal nerve ligation), and degree of
sensitivity was compared between Ab2 treated animals (hashed bars) and control animals
(filled bars). Higher values indicate less sensitivity. Average sensitivity was similar at day
13 (prior to Ab2 administration) but improved at days 14 and 17. Error bars indicate the
standard error of the mean.
shows the analgesic effect of Ab2 and morphine. Pain sensitivity was
assessed by the tail withdrawal time (y-axis, seconds) for animals administered morphine
(open squares), Ab2 (10 mg/kg, filled triangles), or vehicle (negative control, open circles).
Animals developed morphine tolerance and exhibited tail withdrawal times similar to
control animals by day 4. In contrast, Ab2-treated animals exhibited a sustained
improvement in tail withdrawal time over the course of the experiment (to day 7). The
improvement in Ab2-treated animals was statistically significant (p < 0.05 one-way
ANOVA followed by Dunnett’s test, comparison to vehicle, indicated by asterisks). Error
bars indicate the standard error of the mean.
shows the dosage-dependent analgesic effect of Ab2. On day 0
(subsequent to the first tail withdrawal time test), rats were administered antibody Ab2 at a
dosage of 1 mg/kg (filled squares), 3 mg/kg (filled downward-pointing triangles), or 10
mg/kg (filled upward-pointing triangles), or a vehicle (open circles) or negative control
antibody (open squares). The rats’ tail withdrawal time in response to a painful thermal
stimulus was assessed daily (higher times indicate relative insensitivity to pain). Tail
withdrawal time was increased in a dosage-dependent manner by the administration of
Ab2. Asterisks indicate statistically significant increases in tail withdrawal time (p < 0.05
one-way ANOVA followed by Dunnett’s test, comparison to vehicle). Error bars indicate
the standard error of the mean.
shows the analgesic effect of Ab2 in combination with morphine, and
when morphine is withdrawn after the onset of morphine tolerance. On day 0 (subsequent
to the first tail withdrawal time test), rats were administered antibody Ab2 at a dosage of 10
mg/kg (filled squares and filled triangles) or vehicle (open circles). The rats were then
daily administered morphine on days 1 to 10 (filled squares) or only on days 1 to 4 (filled
triangles). The tail withdrawal time initially was greatly increased in the morphine-treated
mice, but decreased on subsequent days indicating morphine tolerance. However, in the
mice from which morphine was withdrawn after day 4, tail withdrawal time increased and
remained elevated between days 5 and 8. Error bars indicate the standard error of the
mean.
shows the effect of Ab2 in a rat model of visceral pain. Visceral pain
was quantified by measuring the colonic distension threshold (higher values indicate less
sensitivity) for naïve animals (open bar) or animals treated with TNBS to provoke chronic
colonic hypersensitivity which either received a negative control antibody (filled bars) or
Ab2 (hashed bars). Hypersensitivity was alleviated by 27% by the Ab2-treated animals,
and distension threshold was significantly improved by administration of Ab2 (p < 0.05
Student’s t-test, comparison to TNBS + Negative control group). Error bars indicate the
standard error of the mean.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Definitions
It is to be understood that this invention is not limited to the particular
methodology, protocols, cell lines, animal species or genera, and reagents described, as
such may vary. It is also to be understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is not intended to limit the scope
of the present invention which will be limited only by the appended claims. As used herein
the singular forms "a", "and", and "the" include plural referents unless the context clearly
dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such
cells and reference to "the protein" includes reference to one or more proteins and
equivalents thereof known to those skilled in the art, and so forth. All technical and
scientific terms used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.
Calcitonin Gene Related Peptide (CGRP): As used herein, CGRP encompasses
not only the following Homo sapiens CGRP-alpha and Homo sapiens CGRP-beta amino
acid sequences available from American Peptides (Sunnyvale CA) and Bachem (Torrance,
CA):
CGRP-alpha: ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF-NH (SEQ ID
NO: 281), wherein the N-terminal phenylalanine is amidated;
CGRP-beta: ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF-NH (SEQ ID
NO: 282), wherein the N-terminal phenylalanine is amidated; but also any membrane-
bound forms of these CGRP amino acid sequences, as well as mutants (mutiens), splice
variants, isoforms, orthologues, homologues and variants of this sequence.
Mating competent yeast species: In the present disclosure this is intended to
broadly encompass any diploid or tetraploid yeast which can be grown in culture. Such
species of yeast may exist in a haploid, diploid, or other polyploid form. The cells of a
given ploidy may, under appropriate conditions, proliferate for an indefinite number of
generations in that form. Diploid cells can also sporulate to form haploid cells. Sequential
mating can result in tetraploid strains through further mating or fusion of diploid strains.
The present disclosure contemplates the use of haploid yeast, as well as diploid or other
polyploid yeast cells produced, for example, by mating or spheroplast fusion.
In one embodiment of the disclosure, the mating competent yeast is a member of
the Saccharomycetaceae family, which includes the genera Arxiozyma; Ascobotryozyma;
Citeromyces; Debaryomyces; Dekkera; Eremothecium; Issatchenkia; Kazachstania;
Kluyveromyces; Kodamaea; Lodderomyces; Pachysolen; Pichia; Saccharomyces;
Saturnispora; Tetrapisispora; Torulaspora; Williopsis; and Zygosaccharomyces. Other
types of yeast potentially useful herein include Yarrowia; Rhodosporidium; Candida;
Hansenula; Filobasium; Sporidiobolus; Bullera; Leucosporidium and Filobasidella.
In a preferred embodiment of the disclosure, the mating competent yeast is a
member of the genus Pichia. In a further preferred embodiment of the disclosure, the
mating competent yeast of the genus Pichia is one of the following species: Pichia
pastoris, Pichia methanolica, and Hansenula polymorpha (Pichia angusta). In a
particularly preferred embodiment of the disclosure, the mating competent yeast of the
genus Pichia is the species Pichia pastoris.
Haploid Yeast Cell: A cell having a single copy of each gene of its normal
genomic (chromosomal) complement.
Polyploid Yeast Cell: A cell having more than one copy of its normal genomic
(chromosomal) complement.
Diploid Yeast Cell: A cell having two copies (alleles) of essentially every gene
of its normal genomic complement, typically formed by the process of fusion (mating) of
two haploid cells.
Tetraploid Yeast Cell: A cell having four copies (alleles) of essentially every
gene of its normal genomic complement, typically formed by the process of fusion
(mating) of two haploid cells. Tetraploids may carry two, three, four or more different
expression cassettes. Such tetraploids might be obtained in S. cerevisiae by selective
mating homozygotic heterothallic a/a and alpha/alpha diploids and in Pichia by sequential
mating of haploids to obtain auxotrophic diploids. For example, a [met his] haploid can be
mated with [ade his] haploid to obtain diploid [his]; and a [met arg] haploid can be mated
with [ade arg] haploid to obtain diploid [arg]; then the diploid [his] x diploid [arg] to obtain
a tetraploid prototroph. It will be understood by those of skill in the art that reference to
the benefits and uses of diploid cells may also apply to tetraploid cells.
Yeast Mating: The process by which two haploid yeast cells naturally fuse to
form one diploid yeast cell.
Meiosis: The process by which a diploid yeast cell undergoes reductive division
to form four haploid spore products. Each spore may then germinate and form a haploid
vegetatively growing cell line.
Selectable Marker: A selectable marker is a gene or gene fragment that confers
a growth phenotype (physical growth characteristic) on a cell receiving that gene as, for
example through a transformation event. The selectable marker allows that cell to survive
and grow in a selective growth medium under conditions in which cells that do not receive
that selectable marker gene cannot grow. Selectable marker genes generally fall into
several types, including positive selectable marker genes such as a gene that confers on a
cell resistance to an antibiotic or other drug, temperature when two temperature sensitive
(“ts”) mutants are crossed or a ts mutant is transformed; negative selectable marker genes
such as a biosynthetic gene that confers on a cell the ability to grow in a medium without a
specific nutrient needed by all cells that do not have that biosynthetic gene, or a
mutagenized biosynthetic gene that confers on a cell inability to grow by cells that do not
have the wild type gene; and the like. Suitable markers include but are not limited to:
ZEO; G418; LYS3; MET1; MET3a; ADE1; ADE3; URA3; and the like.
Expression Vector: These DNA vectors contain elements that facilitate
manipulation for the expression of a foreign protein within the target host cell.
Conveniently, manipulation of sequences and production of DNA for transformation is first
performed in a bacterial host, e.g. E. coli, and usually vectors will include sequences to
facilitate such manipulations, including a bacterial origin of replication and appropriate
bacterial selection marker. Selection markers encode proteins necessary for the survival or
growth of transformed host cells grown in a selective culture medium. Host cells not
transformed with the vector containing the selection gene will not survive in the culture
medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or
other toxins, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not
available from complex media. Exemplary vectors and methods for transformation of
yeast are described, for example, in Burke, D., Dawson, D., & Stearns, T. (2000). Methods
in yeast genetics: a Cold Spring Harbor Laboratory course manual. Plainview, N.Y.: Cold
Spring Harbor Laboratory Press.
Expression vectors for use in the methods described herein will further include
yeast specific sequences, including a selectable auxotrophic or drug marker for identifying
transformed yeast strains. A drug marker may further be used to amplify copy number of
the vector in a yeast host cell.
The polypeptide coding sequence of interest is operably linked to transcriptional
and translational regulatory sequences that provide for expression of the polypeptide in
yeast cells. These vector components may include, but are not limited to, one or more of
the following: an enhancer element, a promoter, and a transcription termination sequence.
Sequences for the secretion of the polypeptide may also be included, e.g. a signal sequence,
and the like. A yeast origin of replication is optional, as expression vectors are often
integrated into the yeast genome. In one embodiment of the disclosure, the polypeptide of
interest is operably linked, or fused, to sequences providing for optimized secretion of the
polypeptide from yeast diploid cells.
Nucleic acids are "operably linked" when placed into a functional relationship
with another nucleic acid sequence. For example, DNA for a signal sequence is operably
linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the
secretion of the polypeptide; a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the sequence. Generally, "operably linked" means
that the DNA sequences being linked are contiguous, and, in the case of a secretory leader,
contiguous and in reading frame. However, enhancers do not have to be contiguous.
Linking is accomplished by ligation at convenient restriction sites or alternatively via a
PCR/recombination method familiar to those skilled in the art (Gateway Technology;
Invitrogen, Carlsbad California). If such sites do not exist, the synthetic oligonucleotide
adapters or linkers are used in accordance with conventional practice.
Promoters are untranslated sequences located upstream (5') to the start codon of
a structural gene (generally within about 100 to 1000 bp) that control the transcription and
translation of particular nucleic acid sequences to which they are operably linked. Such
promoters fall into several classes: inducible, constitutive, and repressible promoters (that
increase levels of transcription in response to absence of a repressor). Inducible promoters
may initiate increased levels of transcription from DNA under their control in response to
some change in culture conditions, e.g., the presence or absence of a nutrient or a change in
temperature.
The yeast promoter fragment may also serve as the site for homologous
recombination and integration of the expression vector into the same site in the yeast
genome; alternatively a selectable marker is used as the site for homologous
recombination. Pichia transformation is described in Cregg et al. (1985) Mol. Cell. Biol.
:3376-3385.
Examples of suitable promoters from Pichia include the AOX1 and promoter
(Cregg et al. (1989) Mol. Cell. Biol. 9:1316-1323); ICL1 promoter (Menendez et al. (2003)
Yeast 20(13):1097-108); glyceraldehydephosphate dehydrogenase promoter (GAP)
(Waterham et al. (1997) Gene 186(1):37-44); and FLD1 promoter (Shen et al. (1998) Gene
216(1):93-102). The GAP promoter is a strong constitutive promoter and the AOX and
FLD1 promoters are inducible.
Other yeast promoters include ADH1, alcohol dehydrogenase II, GAL4, PHO3,
PHO5, Pyk, and chimeric promoters derived therefrom. Additionally, non-yeast promoters
may be used herein such as mammalian, insect, plant, reptile, amphibian, viral, and avian
promoters. Most typically the promoter will comprise a mammalian promoter (potentially
endogenous to the expressed genes) or will comprise a yeast or viral promoter that provides
for efficient transcription in yeast systems.
The polypeptides of interest may be produced recombinantly not only directly,
but also as a fusion polypeptide with a heterologous polypeptide, e.g. a signal sequence or
other polypeptide having a specific cleavage site at the N-terminus of the mature protein or
polypeptide. In general, the signal sequence may be a component of the vector, or it may
be a part of the polypeptide coding sequence that is inserted into the vector. The
heterologous signal sequence selected preferably is one that is recognized and processed
through one of the standard pathways available within the host cell. The S. cerevisiae
alpha factor pre-pro signal has proven effective in the secretion of a variety of recombinant
proteins from P. pastoris. Other yeast signal sequences include the alpha mating factor
signal sequence, the invertase signal sequence, and signal sequences derived from other
secreted yeast polypeptides. Additionally, these signal peptide sequences may be
engineered to provide for enhanced secretion in diploid yeast expression systems. Other
secretion signals of interest also include mammalian signal sequences, which may be
heterologous to the protein being secreted, or may be a native sequence for the protein
being secreted. Signal sequences include pre-peptide sequences, and in some instances
may include propeptide sequences. Many such signal sequences are known in the art,
including the signal sequences found on immunoglobulin chains, e.g., K28 preprotoxin
sequence, PHA-E, FACE, human MCP-1, human serum albumin signal sequences, human
Ig heavy chain, human Ig light chain, and the like. For example, see Hashimoto et. al.
Protein Eng 11(2) 75 (1998); and Kobayashi et. al. Therapeutic Apheresis 2(4) 257 (1998).
Transcription may be increased by inserting a transcriptional activator sequence
into the vector. These activators are cis-acting elements of DNA, usually about from 10 to
300 bp, which act on a promoter to increase its transcription. Transcriptional enhancers are
relatively orientation and position independent, having been found 5' and 3' to the
transcription unit, within an intron, as well as within the coding sequence itself. The
enhancer may be spliced into the expression vector at a position 5' or 3' to the coding
sequence, but is preferably located at a site 5' from the promoter.
Expression vectors used in eukaryotic host cells may also contain sequences
necessary for the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from 3' to the translation termination codon, in
untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of
the mRNA.
Construction of suitable vectors containing one or more of the above-listed
components employs standard ligation techniques or PCR/recombination methods.
Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligated in the form
desired to generate the plasmids required or via recombination methods. For analysis to
confirm correct sequences in plasmids constructed, the ligation mixtures are used to
transform host cells, and successful transformants selected by antibiotic resistance (e.g.
ampicillin or Zeocin) where appropriate. Plasmids from the transformants are prepared,
analyzed by restriction endonuclease digestion and/or sequenced.
As an alternative to restriction and ligation of fragments, recombination methods
based on att sites and recombination enzymes may be used to insert DNA sequences into a
vector. Such methods are described, for example, by Landy (1989) Ann.Rev.Biochem.
58:913-949; and are known to those of skill in the art. Such methods utilize intermolecular
DNA recombination that is mediated by a mixture of lambda and E.coli –encoded
recombination proteins. Recombination occurs between specific attachment (att) sites on
the interacting DNA molecules. For a description of att sites see Weisberg and Landy
(1983) Site-Specific Recombination in Phage Lambda, in Lambda II, Weisberg, ed.(Cold
Spring Harbor, NY:Cold Spring Harbor Press), pp. 211-250. The DNA segments flanking
the recombination sites are switched, such that after recombination, the att sites are hybrid
sequences comprised of sequences donated by each parental vector. The recombination
can occur between DNAs of any topology.
Att sites may be introduced into a sequence of interest by ligating the sequence
of interest into an appropriate vector; generating a PCR product containing att B sites
through the use of specific primers; generating a cDNA library cloned into an appropriate
vector containing att sites; and the like.
Folding, as used herein, refers to the three-dimensional structure of polypeptides
and proteins, where interactions between amino acid residues act to stabilize the structure.
While non-covalent interactions are important in determining structure, usually the proteins
of interest will have intra- and/or intermolecular covalent disulfide bonds formed by two
cysteine residues. For naturally occurring proteins and polypeptides or derivatives and
variants thereof, the proper folding is typically the arrangement that results in optimal
biological activity, and can conveniently be monitored by assays for activity, e.g. ligand
binding, enzymatic activity, etc.
In some instances, for example where the desired product is of synthetic origin,
assays based on biological activity will be less meaningful. The proper folding of such
molecules may be determined on the basis of physical properties, energetic considerations,
modeling studies, and the like.
The expression host may be further modified by the introduction of sequences
encoding one or more enzymes that enhance folding and disulfide bond formation, i.e.
foldases, chaperonins, etc. Such sequences may be constitutively or inducibly expressed in
the yeast host cell, using vectors, markers, etc. as known in the art. Preferably the
sequences, including transcriptional regulatory elements sufficient for the desired pattern of
expression, are stably integrated in the yeast genome through a targeted methodology.
For example, the eukaryotic PDI is not only an efficient catalyst of protein
cysteine oxidation and disulfide bond isomerization, but also exhibits chaperone activity.
Co-expression of PDI can facilitate the production of active proteins having multiple
disulfide bonds. Also of interest is the expression of BIP (immunoglobulin heavy chain
binding protein); cyclophilin; and the like. In one embodiment of the disclosure, each of
the haploid parental strains expresses a distinct folding enzyme, e.g. one strain may express
BIP, and the other strain may express PDI or combinations thereof.
The terms "desired protein" or "desired antibody" are used interchangeably and
refer generally to a parent antibody specific to a target, i.e., CGRP or a chimeric or
humanized antibody or a binding portion thereof derived therefrom as described herein.
The term “antibody” is intended to include any polypeptide chain-containing molecular
structure with a specific shape that fits to and recognizes an epitope, where one or more
non-covalent binding interactions stabilize the complex between the molecular structure
and the epitope. The archetypal antibody molecule is the immunoglobulin, and all types of
immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human, rodent,
rabbit, cow, sheep, pig, dog, other mammals, chicken, other avians, etc., are considered to
be “antibodies.” A preferred source for producing antibodies useful as starting material
according to the present disclosure is rabbits. Numerous antibody coding sequences have
been described; and others may be raised by methods well-known in the art. Examples
thereof include chimeric antibodies, human antibodies and other non-human mammalian
antibodies, humanized antibodies, single chain antibodies (such as scFvs), camelbodies,
nanobodies, IgNAR (single-chain antibodies derived from sharks), small-modular
immunopharmaceuticals (SMIPs), and antibody fragments such as Fabs, Fab', F(ab') and
the like. See Streltsov VA, et al., Structure of a shark IgNAR antibody variable domain
and modeling of an early-developmental isotype, Protein Sci. 2005 Nov;14(11):2901-9.
Epub 2005 Sep 30; Greenberg AS, et al., A new antigen receptor gene family that
undergoes rearrangement and extensive somatic diversification in sharks, Nature. 1995 Mar
9;374(6518):168-73; Nuttall SD, et al., Isolation of the new antigen receptor from
wobbegong sharks, and use as a scaffold for the display of protein loop libraries, Mol
Immunol. 2001 Aug;38(4):313-26; Hamers-Casterman C, et al., Naturally occurring
antibodies devoid of light chains, Nature. 1993 Jun 3;363(6428):446-8; Gill DS, et al.,
Biopharmaceutical drug discovery using novel protein scaffolds, Curr Opin Biotechnol.
2006 Dec; 17(6):653-8. Epub 2006 Oct 19.
For example, antibodies or antigen binding fragments may be produced by
genetic engineering. In this technique, as with other methods, antibody-producing cells are
sensitized to the desired antigen or immunogen. The messenger RNA isolated from
antibody producing cells is used as a template to make cDNA using PCR amplification. A
library of vectors, each containing one heavy chain gene and one light chain gene retaining
the initial antigen specificity, is produced by insertion of appropriate sections of the
amplified immunoglobulin cDNA into the expression vectors. A combinatorial library is
constructed by combining the heavy chain gene library with the light chain gene library.
This results in a library of clones which co-express a heavy and light chain (resembling the
Fab fragment or antigen binding fragment of an antibody molecule). The vectors that carry
these genes are co-transfected into a host cell. When antibody gene synthesis is induced in
the transfected host, the heavy and light chain proteins self-assemble to produce active
antibodies that can be detected by screening with the antigen or immunogen.
Antibody coding sequences of interest include those encoded by native
sequences, as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are
not identical in sequence to the disclosed nucleic acids, and variants thereof. Variant
polypeptides can include amino acid (aa) substitutions, additions or deletions. The amino
acid substitutions can be conservative amino acid substitutions or substitutions to eliminate
non-essential amino acids, such as to alter a glycosylation site, or to minimize misfolding
by substitution or deletion of one or more cysteine residues that are not necessary for
function. Variants can be designed so as to retain or have enhanced biological activity of a
particular region of the protein (e.g., a functional domain, catalytic amino acid residues,
etc). Variants also include fragments of the polypeptides disclosed herein, particularly
biologically active fragments and/or fragments corresponding to functional domains.
Techniques for in vitro mutagenesis of cloned genes are known. Also included in the
present disclosure are polypeptides that have been modified using ordinary molecular
biological techniques so as to improve their resistance to proteolytic degradation or to
optimize solubility properties or to render them more suitable as a therapeutic agent.
Chimeric antibodies may be made by recombinant means by combining the
variable light and heavy chain regions (V and V ), obtained from antibody producing cells
of one species with the constant light and heavy chain regions from another. Typically
chimeric antibodies utilize rodent or rabbit variable regions and human constant regions, in
order to produce an antibody with predominantly human domains. The production of such
chimeric antibodies is well known in the art, and may be achieved by standard means (as
described, e.g., in U.S. Patent No. 5,624,659, incorporated herein by reference in its
entirety). It is further contemplated that the human constant regions of chimeric antibodies
described herein may be selected from IgG1, IgG2, IgG3, and IgG4 constant regions.
Humanized antibodies are engineered to contain even more human-like
immunoglobulin domains, and incorporate only the complementarity-determining regions
of the animal-derived antibody. This is accomplished by carefully examining the sequence
of the hyper-variable loops of the variable regions of the monoclonal antibody, and fitting
them to the structure of the human antibody chains. Although facially complex, the
process is straightforward in practice. See, e.g., U.S. Patent No. 6,187,287, incorporated
fully herein by reference.
In addition to entire immunoglobulins (or their recombinant counterparts),
immunoglobulin fragments comprising the epitope binding site (e.g., Fab’, F(ab’) , or other
fragments) may be synthesized. “Fragment,” or minimal immunoglobulins may be
designed utilizing recombinant immunoglobulin techniques. For instance “Fv”
immunoglobulins for use in the present disclosure may be produced by synthesizing a
fused variable light chain region and a variable heavy chain region. Combinations of
antibodies are also of interest, e.g. diabodies, which comprise two distinct Fv specificities.
In another embodiment of the disclosure, SMIPs (small molecule
immunopharmaceuticals), camelbodies, nanobodies, and IgNAR are encompassed by
immunoglobulin fragments.
Immunoglobulins and fragments thereof may be modified post-translationally,
e.g. to add effector moieties such as chemical linkers, detectable moieties, such as
fluorescent dyes, enzymes, toxins, substrates, bioluminescent materials, radioactive
materials, chemiluminescent moieties and the like, or specific binding moieties, such as
streptavidin, avidin, or biotin, and the like may be utilized in the methods and compositions
of the present disclosure. Examples of additional effector molecules are provided infra.
A polynucleotide sequence "corresponds" to a polypeptide sequence if
translation of the polynucleotide sequence in accordance with the genetic code yields the
polypeptide sequence (i.e., the polynucleotide sequence "encodes" the polypeptide
sequence), one polynucleotide sequence "corresponds" to another polynucleotide sequence
if the two sequences encode the same polypeptide sequence.
A "heterologous" region or domain of a DNA construct is an identifiable
segment of DNA within a larger DNA molecule that is not found in association with the
larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene,
the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA
in the genome of the source organism. Another example of a heterologous region is a
construct where the coding sequence itself is not found in nature (e.g., a cDNA where the
genomic coding sequence contains introns, or synthetic sequences having codons different
than the native gene). Allelic variations or naturally-occurring mutational events do not
give rise to a heterologous region of DNA as defined herein.
A "coding sequence" is an in-frame sequence of codons that (in view of the
genetic code) correspond to or encode a protein or peptide sequence. Two coding
sequences correspond to each other if the sequences or their complementary sequences
encode the same amino acid sequences. A coding sequence in association with appropriate
regulatory sequences may be transcribed and translated into a polypeptide. A
polyadenylation signal and transcription termination sequence will usually be located 3' to
the coding sequence. A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a downstream (3'
direction) coding sequence. Promoter sequences typically contain additional sites for
binding of regulatory molecules (e.g., transcription factors) which affect the transcription
of the coding sequence. A coding sequence is "under the control" of the promoter sequence
or "operatively linked" to the promoter when RNA polymerase binds the promoter
sequence in a cell and transcribes the coding sequence into mRNA, which is then in turn
translated into the protein encoded by the coding sequence.
Vectors are used to introduce a foreign substance, such as DNA, RNA or
protein, into an organism or host cell. Typical vectors include recombinant viruses (for
polynucleotides) and liposomes (for polypeptides). A "DNA vector" is a replicon, such as
plasmid, phage or cosmid, to which another polynucleotide segment may be attached so as
to bring about the replication of the attached segment. An "expression vector" is a DNA
vector which contains regulatory sequences which will direct polypeptide synthesis by an
appropriate host cell. This usually means a promoter to bind RNA polymerase and initiate
transcription of mRNA, as well as ribosome binding sites and initiation signals to direct
translation of the mRNA into a polypeptide(s). Incorporation of a polynucleotide sequence
into an expression vector at the proper site and in correct reading frame, followed by
transformation of an appropriate host cell by the vector, enables the production of a
polypepide encoded by said polynucleotide sequence.
"Amplification" of polynucleotide sequences is the in vitro production of
multiple copies of a particular nucleic acid sequence. The amplified sequence is usually in
the form of DNA. A variety of techniques for carrying out such amplification are
described in a review article by Van Brunt (1990, Bio/Technol., 8(4):291-294). Polymerase
chain reaction or PCR is a prototype of nucleic acid amplification, and use of PCR herein
should be considered exemplary of other suitable amplification techniques.
The general structure of antibodies in vertebrates now is well understood
(Edelman, G. M., Ann. N.Y. Acad. Sci., 190: 5 (1971)). Antibodies consist of two
identical light polypeptide chains of molecular weight approximately 23,000 daltons (the
“light chain”), and two identical heavy chains of molecular weight 53,000-70,000 (the
“heavy chain”). The four chains are joined by disulfide bonds in a “Y” configuration
wherein the light chains bracket the heavy chains starting at the mouth of the “Y”
configuration. The “branch” portion of the “Y” configuration is designated the F region;
the stem portion of the “Y” configuration is designated the FC region. The amino acid
sequence orientation runs from the N-terminal end at the top of the “Y” configuration to
the C-terminal end at the bottom of each chain. The N-terminal end possesses the variable
region having specificity for the antigen that elicited it, and is approximately 100 amino
acids in length, there being slight variations between light and heavy chain and from
antibody to antibody.
The variable region is linked in each chain to a constant region that extends the
remaining length of the chain and that within a particular class of antibody does not vary
with the specificity of the antibody (i.e., the antigen eliciting it). There are five known
major classes of constant regions that determine the class of the immunoglobulin molecule
(IgG, IgM, IgA, IgD, and IgE corresponding to γ, μ, α, δ, and ε (gamma, mu, alpha, delta,
or epsilon) heavy chain constant regions). The constant region or class determines
subsequent effector function of the antibody, including activation of complement (Kabat,
E. A., Structural Concepts in Immunology and Immunochemistry, 2nd Ed., p. 413-436,
Holt, Rinehart, Winston (1976)), and other cellular responses (Andrews, D. W., et al.,
Clinical Immunobiology, pp 1-18, W. B. Sanders (1980); Kohl, S., et al., Immunology, 48:
187 (1983)); while the variable region determines the antigen with which it will react.
Light chains are classified as either κ (kappa) or λ (lambda). Each heavy chain class can be
prepared with either kappa or lambda light chain. The light and heavy chains are
covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded
to each other by covalent disulfide linkages when the immunoglobulins are generated either
by hybridomas or by B cells.
The expression “variable region” or “VR” refers to the domains within each pair
of light and heavy chains in an antibody that are involved directly in binding the antibody
to the antigen. Each heavy chain has at one end a variable domain (V ) followed by a
number of constant domains. Each light chain has a variable domain (V ) at one end and a
constant domain at its other end; the constant domain of the light chain is aligned with the
first constant domain of the heavy chain, and the light chain variable domain is aligned
with the variable domain of the heavy chain.
The expressions “complementarity determining region,” “hypervariable region,”
or “CDR” refer to one or more of the hyper-variable or complementarity determining
regions (CDRs) found in the variable regions of light or heavy chains of an antibody (See
Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National Institutes of
Health, Bethesda, Md., (1987)). These expressions include the hypervariable regions as
defined by Kabat et al. (“Sequences of Proteins of Immunological Interest,” Kabat E., et
al., US Dept. of Health and Human Services, 1983) or the hypervariable loops in 3-
dimensional structures of antibodies (Chothia and Lesk, J Mol. Biol. 196 901-917 (1987)).
The CDRs in each chain are held in close proximity by framework regions and, with the
CDRs from the other chain, contribute to the formation of the antigen binding site. Within
the CDRs there are select amino acids that have been described as the selectivity
determining regions (SDRs) which represent the critical contact residues used by the CDR
in the antibody-antigen interaction (Kashmiri, S., Methods, 36:25-34 (2005)).
The expressions “framework region” or “FR” refer to one or more of the
framework regions within the variable regions of the light and heavy chains of an antibody
(See Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National
Institutes of Health, Bethesda, Md., (1987)). These expressions include those amino acid
sequence regions interposed between the CDRs within the variable regions of the light and
heavy chains of an antibody.
Anti-CGRP Antibodies and Binding Fragments Thereof Having Binding Activity for
CGRP
Antibody Ab1
In one embodiment, the disclosure includes chimeric antibodies having binding
specificity to CGRP and possessing a variable light chain sequence comprising the
sequence set forth below:
QVLTQTASPVSAAVGSTVTINCQASQSVYDNNYLAWYQQKPGQPPKQLIYSTSTL
ASGVSSRFKGSGSGTQFTLTISDLECADAATYYCLGSYDCSSGDCFVFGGGTEVVV
KR (SEQ ID NO: 1).
The disclosure also includes chimeric antibodies having binding specificity to
CGRP and possessing a light chain sequence comprising the sequence set forth below:
QVLTQTASPVSAAVGSTVTINCQASQSVYDNNYLAWYQQKPGQPPKQLIYSTSTL
ASGVSSRFKGSGSGTQFTLTISDLECADAATYYCLGSYDCSSGDCFVFGGGTEVVV
KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 2).
The disclosure further includes chimeric antibodies having binding specificity to
CGRP and possessing a variable heavy chain sequence comprising the sequence set forth
below:
QSLEESGGRLVTPGTPLTLTCTVSGLDLSSYYMQWVRQAPGKGLEWIGVIGINDNT
YYASWAKGRFTISRASSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSS
(SEQ ID NO: 3).
The disclosure also includes chimeric antibodies having binding specificity to
CGRP and possessing a heavy chain sequence comprising the sequence set forth below:
QSLEESGGRLVTPGTPLTLTCTVSGLDLSSYYMQWVRQAPGKGLEWIGVIGINDNT
YYASWAKGRFTISRASSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO: 4).
The disclosure further contemplates antibodies comprising one or more of the
polypeptide sequences of SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 1 or the light chain sequence of SEQ
ID NO: 2, and/or one or more of the polypeptide sequences of SEQ ID NO: 8; SEQ ID NO:
9; and SEQ ID NO: 10 which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 3 or
the heavy chain sequence of SEQ ID NO: 4, or combinations of these polypeptide
sequences. In another embodiment of the present disclosure, the antibodies described
herein or fragments thereof comprise, or alternatively consist of, combinations of one or
more of the CDRs, the variable heavy and variable light chain sequences, and the heavy
and light chain sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment of the present disclosure, antibody
fragments described herein comprise, or alternatively consist of, the polypeptide sequence
of SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment described herein, antibody
fragments may comprise, or alternatively consist of, the polypeptide sequence of SEQ ID
NO: 3 or SEQ ID NO: 4.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 1 or the light chain sequence of SEQ
ID NO: 2.
In a further embodimentdescribed are fragments of the antibody having binding
specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide
sequences of SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
heavy chain sequence of SEQ ID NO: 3 or the heavy chain sequence of SEQ ID NO: 4.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment described herein,
fragments of the antibodies having binding specificity to CGRP comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 1; the variable heavy chain region of SEQ ID
NO: 3; the complementarity-determining regions (SEQ ID NO: 5; SEQ ID NO: 6; and SEQ
ID NO: 7) of the variable light chain region of SEQ ID NO: 1; and the complementarity-
determining regions (SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10) of the variable
heavy chain region of SEQ ID NO: 3.
In a particularly preferred embodiment described herein, the chimeric anti-
CGRP antibody is Ab1, comprising, or alternatively consisting of, SEQ ID NO: 2 and SEQ
ID NO: 4, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment described herein, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab1, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 1 and the variable heavy chain
sequence of SEQ ID NO: 3. This embodiment described herein further contemplates
additions, deletions, and variants of SEQ ID NO: 1 and/or SEQ ID NO: 3 in said Fab while
retaining binding specificity for CGRP.
In one embodiment described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab1. In another embodiment described herein, anti-
CGRP antibodies such as Ab1 or Fab fragments thereof may be produced via expression in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab2
In one embodiment, the present disclosure includes humanized antibodies having
binding specificity to CGRP and possessing a variable light chain sequence comprising the
sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCFVFGGGTKVEIK
R (SEQ ID NO: 11).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a light chain sequence comprising the sequence set
forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 12).
The present disclosure further includes humanized antibodies having binding
specificity to CGRP and possessing a variable heavy chain sequence comprising the
sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGKGLEWVGVIGIN
DNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSS (SEQ ID NO: 13).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a heavy chain sequence comprising the sequence set
forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGKGLEWVGVIGIN
DNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 14).
The present disclosure further contemplates antibodies comprising one or more
of the polypeptide sequences of SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 17
which correspond to the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 11 or the light chain sequence
of SEQ ID NO: 12, and/or one or more of the polypeptide sequences of SEQ ID NO: 18;
SEQ ID NO: 19; and SEQ ID NO: 20 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 13 or the heavy chain sequence of SEQ ID NO: 14, or combinations of
these polypeptide sequences. In another embodiment described herein, the antibodies or
fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment described herein, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 11 or SEQ
ID NO: 12. In another embodiment described herein, antibody fragments comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 17 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 11 or the light chain sequence of SEQ
ID NO: 12.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 18; SEQ ID NO: 19; and SEQ ID NO: 20 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable heavy chain sequence of SEQ ID NO: 13 or the heavy chain sequence of
SEQ ID NO: 14.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment described herein,
fragments of the antibodies having binding specificity to CGRP comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 11; the variable heavy chain region of SEQ ID
NO: 13; the complementarity-determining regions (SEQ ID NO: 15; SEQ ID NO: 16; and
SEQ ID NO: 17) of the variable light chain region of SEQ ID NO: 11; and the
complementarity-determining regions (SEQ ID NO: 18; SEQ ID NO: 19; and SEQ ID NO:
) of the variable heavy chain region of SEQ ID NO: 13.
In a particularly preferred embodiment described herein, the humanized anti-
CGRP antibody is Ab2, comprising, or alternatively consisting of, SEQ ID NO: 12 and
SEQ ID NO: 14, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment described herein, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab2, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 11 and the variable heavy chain
sequence of SEQ ID NO: 13. This embodiment described herein further contemplates
additions, deletions, and variants of SEQ ID NO: 11 and/or SEQ ID NO: 13 in said Fab
while retaining binding specificity for CGRP.
In one embodiment described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab2. In another embodiment described herein, anti-
CGRP antibodies such as Ab2 or Fab fragments thereof may be produced via expression in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab3
In one embodiment, the present disclosure includes humanized antibodies having
binding specificity to CGRP and possessing a variable light chain sequence comprising the
sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCFVFGGGTKVEIK
R (SEQ ID NO: 21).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a light chain sequence comprising the sequence set
forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 22).
The present disclosure further includes humanized antibodies having binding
specificity to CGRP and possessing a variable heavy chain sequence comprising the
sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGKGLEWVGVIGIN
DNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSS (SEQ ID NO: 23).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a heavy chain sequence comprising the sequence set
forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGKGLEWVGVIGIN
DNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 24).
The present disclosure further contemplates antibodies comprising one or more
of the polypeptide sequences of SEQ ID NO: 25; SEQ ID NO: 26; and SEQ ID NO: 27
which correspond to the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 21 or the light chain sequence
of SEQ ID NO: 22, and/or one or more of the polypeptide sequences of SEQ ID NO: 28;
SEQ ID NO: 29; and SEQ ID NO: 30 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 23 or the heavy chain sequence of SEQ ID NO: 24, or combinations of
these polypeptide sequences. In another embodiment described herein, the antibodies or
fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment described herein, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 21 or SEQ
ID NO: 22. In another embodiment described herein, antibody fragments comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 23 or SEQ ID NO: 24.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 25; SEQ ID NO: 26; and SEQ ID NO: 27 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 21 or the light chain sequence of SEQ
ID NO: 22.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 28; SEQ ID NO: 29; and SEQ ID NO: 30 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable heavy chain sequence of SEQ ID NO: 23 or the heavy chain sequence of
SEQ ID NO: 24.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment described herein,
fragments of the antibodies having binding specificity to CGRP comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 21; the variable heavy chain region of SEQ ID
NO: 23; the complementarity-determining regions (SEQ ID NO: 25; SEQ ID NO: 26; and
SEQ ID NO: 27) of the variable light chain region of SEQ ID NO: 21; and the
complementarity-determining regions (SEQ ID NO: 28; SEQ ID NO: 29; and SEQ ID NO:
) of the variable heavy chain region of SEQ ID NO: 23.
In a particularly preferred embodiment described herein, the chimeric anti-
CGRP antibody is Ab3, comprising, or alternatively consisting of, SEQ ID NO: 22 and
SEQ ID NO: 24, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment described herein, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab3, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 21 and the variable heavy chain
sequence of SEQ ID NO: 23. This embodiment described herein further contemplates
additions, deletions, and variants of SEQ ID NO: 21 and/or SEQ ID NO: 23 in said Fab
while retaining binding specificity for CGRP.
In one embodiment described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab3. In another embodiment described herein, anti-
CGRP antibodies such as Ab3 or Fab fragments thereof may be produced via expression in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab4
In one embodiment, the present disclosure includes chimeric antibodies having
binding specificity to CGRP and possessing a variable light chain sequence comprising the
sequence set forth below:
QVLTQTPSPVSAAVGSTVTINCQASQSVYHNTYLAWYQQKPGQPPKQLIYDASTL
ASGVPSRFSGSGSGTQFTLTISGVQCNDAAAYYCLGSYDCTNGDCFVFGGGTEVV
VKR (SEQ ID NO: 31).
The present disclosure also includes chimeric antibodies having binding
specificity to CGRP and possessing a light chain sequence comprising the sequence set
forth below:
QVLTQTPSPVSAAVGSTVTINCQASQSVYHNTYLAWYQQKPGQPPKQLIYDASTL
ASGVPSRFSGSGSGTQFTLTISGVQCNDAAAYYCLGSYDCTNGDCFVFGGGTEVV
VKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 32).
The present disclosure further includes chimeric antibodies having binding
specificity to CGRP and possessing a variable heavy chain sequence comprising the
sequence set forth below:
QSLEESGGRLVTPGTPLTLTCSVSGIDLSGYYMNWVRQAPGKGLEWIGVIGINGAT
YYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSS
(SEQ ID NO: 33).
The present disclosure also includes chimeric antibodies having binding
specificity to CGRP and possessing a heavy chain sequence comprising the sequence set
forth below:
QSLEESGGRLVTPGTPLTLTCSVSGIDLSGYYMNWVRQAPGKGLEWIGVIGINGAT
YYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO: 34).
The present disclosure further contemplates antibodies comprising one or more
of the polypeptide sequences of SEQ ID NO: 35; SEQ ID NO: 36; and SEQ ID NO: 37
which correspond to the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 31 or the light chain sequence
of SEQ ID NO: 32, and/or one or more of the polypeptide sequences of SEQ ID NO: 38;
SEQ ID NO: 39; and SEQ ID NO: 40 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 33 or the heavy chain sequence of SEQ ID NO: 34, or combinations of
these polypeptide sequences. In another embodiment described herein, the antibodies or
fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment described herein, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 31 or SEQ
ID NO: 32. In another embodiment described herein, antibody fragments comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 33 or SEQ ID NO: 34.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 35; SEQ ID NO: 36; and SEQ ID NO: 37 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 31 or the light chain sequence of SEQ
ID NO: 32.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 38; SEQ ID NO: 39; and SEQ ID NO: 40 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable heavy chain sequence of SEQ ID NO: 33 or the heavy chain sequence of
SEQ ID NO: 34.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment described herein,
fragments of the antibodies having binding specificity to CGRP comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 31; the variable heavy chain region of SEQ ID
NO: 33; the complementarity-determining regions (SEQ ID NO: 35; SEQ ID NO: 36; and
SEQ ID NO: 37) of the variable light chain region of SEQ ID NO: 31; and the
complementarity-determining regions (SEQ ID NO: 38; SEQ ID NO: 39; and SEQ ID NO:
40) of the variable heavy chain region of SEQ ID NO: 33.
In a particularly preferred embodiment described herein, the humanized anti-
CGRP antibody is Ab4, comprising, or alternatively consisting of, SEQ ID NO: 32 and
SEQ ID NO: 34, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment described herein, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab4, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 31 and the variable heavy chain
sequence of SEQ ID NO: 33. This embodiment described herein further contemplates
additions, deletions, and variants of SEQ ID NO: 31 and/or SEQ ID NO: 33 in said Fab
while retaining binding specificity for CGRP.
In one embodiment described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab4. In another embodiment described herein, anti-
CGRP antibodies such as Ab4 or Fab fragments thereof may be produced via expression in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab5
In one embodiment, the present disclosure includes humanized antibodies having
binding specificity to CGRP and possessing a variable light chain sequence comprising the
sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIK
R (SEQ ID NO: 41).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a light chain sequence comprising the sequence set
forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 42).
The present disclosure further includes humanized antibodies having binding
specificity to CGRP and possessing a variable heavy chain sequence comprising the
sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGIN
GATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSS (SEQ ID NO: 43).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a heavy chain sequence comprising the sequence set
forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGIN
GATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 44).
The present disclosure further contemplates antibodies comprising one or more
of the polypeptide sequences of SEQ ID NO: 45; SEQ ID NO: 46; and SEQ ID NO: 47
which correspond to the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 41 or the light chain sequence
of SEQ ID NO: 42, and/or one or more of the polypeptide sequences of SEQ ID NO: 48;
SEQ ID NO: 49; and SEQ ID NO: 50 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 43 or the heavy chain sequence of SEQ ID NO: 44, or combinations of
these polypeptide sequences. In another embodiment described herein, the antibodies or
fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment described herein, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 41 or SEQ
ID NO: 42. In another embodiment described herein, antibody fragments comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 43 or SEQ ID NO: 44.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 45; SEQ ID NO: 46; and SEQ ID NO: 47 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 41 or the light chain sequence of SEQ
ID NO: 42.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 48; SEQ ID NO: 49; and SEQ ID NO: 50 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable heavy chain sequence of SEQ ID NO: 43 or the heavy chain sequence of
SEQ ID NO: 44.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment described herein,
fragments of the antibodies having binding specificity to CGRP comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 41; the variable heavy chain region of SEQ ID
NO: 43; the complementarity-determining regions (SEQ ID NO: 45; SEQ ID NO: 46; and
SEQ ID NO: 47) of the variable light chain region of SEQ ID NO: 41; and the
complementarity-determining regions (SEQ ID NO: 48; SEQ ID NO: 49; and SEQ ID NO:
50) of the variable heavy chain region of SEQ ID NO: 43.
In a particularly preferred embodiment described herein, the chimeric anti-
CGRP antibody is Ab5, comprising, or alternatively consisting of, SEQ ID NO: 42 and
SEQ ID NO: 44, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment described herein, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab5, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 41 and the variable heavy chain
sequence of SEQ ID NO: 43. This embodiment described herein further contemplates
additions, deletions, and variants of SEQ ID NO: 41 and/or SEQ ID NO: 43 in said Fab
while retaining binding specificity for CGRP.
In one embodiment described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab5. In another embodiment described herein, anti-
CGRP antibodies such as Ab5 or Fab fragments thereof may be produced via expression in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab6
In one embodiment, the present disclosure includes humanized antibodies having
binding specificity to CGRP and possessing a variable light chain sequence comprising the
sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIK
R (SEQ ID NO: 51).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a light chain sequence comprising the sequence set
forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 52).
The present disclosure further includes humanized antibodies having binding
specificity to CGRP and possessing a variable heavy chain sequence comprising the
sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGIN
GATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSS (SEQ ID NO: 53).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a heavy chain sequence comprising the sequence set
forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGIN
GATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 54).
The present disclosure further contemplates antibodies comprising one or more
of the polypeptide sequences of SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57
which correspond to the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 51 or the light chain sequence
of SEQ ID NO: 52, and/or one or more of the polypeptide sequences of SEQ ID NO: 58;
SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 54, or combinations of
these polypeptide sequences. In another embodiment described herein, the antibodies or
fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment described herein, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 51 or SEQ
ID NO: 52. In another embodiment described herein, antibody fragments comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 53 or SEQ ID NO: 54.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 51 or the light chain sequence of SEQ
ID NO: 52.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable heavy chain sequence of SEQ ID NO: 53 or the heavy chain sequence of
SEQ ID NO: 54.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment described herein,
fragments of the antibodies having binding specificity to CGRP comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 51; the variable heavy chain region of SEQ ID
NO: 53; the complementarity-determining regions (SEQ ID NO: 55; SEQ ID NO: 56; and
SEQ ID NO: 57) of the variable light chain region of SEQ ID NO: 51; and the
complementarity-determining regions (SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO:
60) of the variable heavy chain region of SEQ ID NO: 53.
In a particularly preferred embodiment described herein, the humanized anti-
CGRP antibody is Ab6, comprising, or alternatively consisting of, SEQ ID NO: 52 and
SEQ ID NO: 54, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment described herein, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab6, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 51 and the variable heavy chain
sequence of SEQ ID NO: 53. This embodiment described herein further contemplates
additions, deletions, and variants of SEQ ID NO: 51 and/or SEQ ID NO: 53 in said Fab
while retaining binding specificity for CGRP.
In one embodiment described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab6. In another embodiment described herein, anti-
CGRP antibodies such as Ab6 or Fab fragments thereof may be produced via expression in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab7
In one embodiment, the present disclosure includes chimeric antibodies having
binding specificity to CGRP and possessing a variable light chain sequence comprising the
sequence set forth below:
QVLTQTASPVSAAVGSTVTINCQASQSVYNYNYLAWYQQKPGQPPKQLIYSTSTL
ASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSTGDCFVFGGGTEVV
VKR (SEQ ID NO: 61).
The present disclosure also includes chimeric antibodies having binding
specificity to CGRP and possessing a light chain sequence comprising the sequence set
forth below:
QVLTQTASPVSAAVGSTVTINCQASQSVYNYNYLAWYQQKPGQPPKQLIYSTSTL
ASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSTGDCFVFGGGTEVV
VKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 62).
The present disclosure further includes chimeric antibodies having binding
specificity to CGRP and possessing a variable heavy chain sequence comprising the
sequence set forth below:
QEQLKESGGRLVTPGTSLTLTCTVSGIDLSNHYMQWVRQAPGKGLEWIGVVGING
RTYYASWAKGRFTISRTSSTTVDLKMTRLTTEDTATYFCARGDIWGPGTLVTVSS
(SEQ ID NO: 63).
The present disclosure also includes chimeric antibodies having binding
specificity to CGRP and possessing a heavy chain sequence comprising the sequence set
forth below:
QEQLKESGGRLVTPGTSLTLTCTVSGIDLSNHYMQWVRQAPGKGLEWIGVVGING
RTYYASWAKGRFTISRTSSTTVDLKMTRLTTEDTATYFCARGDIWGPGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ
ID NO: 64).
The present disclosure further contemplates antibodies comprising one or more
of the polypeptide sequences of SEQ ID NO: 65; SEQ ID NO: 66; and SEQ ID NO: 67
which correspond to the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 61 or the light chain sequence
of SEQ ID NO: 62, and/or one or more of the polypeptide sequences of SEQ ID NO: 68;
SEQ ID NO: 69; and SEQ ID NO: 70 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 63 or the heavy chain sequence of SEQ ID NO: 64, or combinations of
these polypeptide sequences. In another embodiment described herein, the antibodies or
fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment described herein, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 61 or SEQ
ID NO: 62. In another embodiment described herein, antibody fragments comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 63 or SEQ ID NO: 64.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 65; SEQ ID NO: 66; and SEQ ID NO: 67 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 61 or the light chain sequence of SEQ
ID NO: 62.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 68; SEQ ID NO: 69; and SEQ ID NO: 70 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable heavy chain sequence of SEQ ID NO: 63 or the heavy chain sequence of
SEQ ID NO: 64.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment described herein,
fragments of the antibodies having binding specificity to CGRP comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 61; the variable heavy chain region of SEQ ID
NO: 63; the complementarity-determining regions (SEQ ID NO: 65; SEQ ID NO: 66; and
SEQ ID NO: 67) of the variable light chain region of SEQ ID NO: 61; and the
complementarity-determining regions (SEQ ID NO: 68; SEQ ID NO: 69; and SEQ ID NO:
70) of the variable heavy chain region of SEQ ID NO: 63.
In a particularly preferred embodiment described herein, the chimeric anti-
CGRP antibody is Ab7, comprising, or alternatively consisting of, SEQ ID NO: 62 and
SEQ ID NO: 64, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment described herein, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab7, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 61 and the variable heavy chain
sequence of SEQ ID NO: 63. This embodiment described herein further contemplates
additions, deletions, and variants of SEQ ID NO: 61 and/or SEQ ID NO: 63 in said Fab
while retaining binding specificity for CGRP.
In one embodiment described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab7. In another embodiment described herein, anti-
CGRP antibodies such as Ab7 or Fab fragments thereof may be produced via expression in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab8
In one embodiment, the present disclosure includes humanized antibodies having
binding specificity to CGRP and possessing a variable light chain sequence comprising the
sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYNYNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSTGDCFVFGGGTKVEIK
R (SEQ ID NO: 71).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a light chain sequence comprising the sequence set
forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYNYNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSTGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 72).
The present disclosure further includes humanized antibodies having binding
specificity to CGRP and possessing a variable heavy chain sequence comprising the
sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDLSNHYMQWVRQAPGKGLEWVGVVGIN
GRTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSS (SEQ ID NO: 73).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a heavy chain sequence comprising the sequence set
forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDLSNHYMQWVRQAPGKGLEWVGVVGIN
GRTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 74).
The present disclosure further contemplates antibodies comprising one or more
of the polypeptide sequences of SEQ ID NO: 75; SEQ ID NO: 76; and SEQ ID NO: 77
which correspond to the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 71 or the light chain sequence
of SEQ ID NO: 72, and/or one or more of the polypeptide sequences of SEQ ID NO: 78;
SEQ ID NO: 79; and SEQ ID NO: 80 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 73 or the heavy chain sequence of SEQ ID NO: 74, or combinations of
these polypeptide sequences. In another embodiment described herein, the antibodies or
fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment described herein, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 71 or SEQ
ID NO: 72. In another embodiment described herein, antibody fragments comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 73 or SEQ ID NO: 74.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 75; SEQ ID NO: 76; and SEQ ID NO: 77 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 71 or the light chain sequence of SEQ
ID NO: 72.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 78; SEQ ID NO: 79; and SEQ ID NO: 80 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable heavy chain sequence of SEQ ID NO: 73 or the heavy chain sequence of
SEQ ID NO: 74.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment described herein,
fragments of the antibodies having binding specificity to CGRP comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 71; the variable heavy chain region of SEQ ID
NO: 73; the complementarity-determining regions (SEQ ID NO: 75; SEQ ID NO: 76; and
SEQ ID NO: 77) of the variable light chain region of SEQ ID NO: 71; and the
complementarity-determining regions (SEQ ID NO: 78; SEQ ID NO: 79; and SEQ ID NO:
80) of the variable heavy chain region of SEQ ID NO: 73.
In a particularly preferred embodiment described herein, the humanized anti-
CGRP antibody is Ab8, comprising, or alternatively consisting of, SEQ ID NO: 72 and
SEQ ID NO: 74, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment described herein, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab8, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 71 and the variable heavy chain
sequence of SEQ ID NO: 73. This embodiment described herein further contemplates
additions, deletions, and variants of SEQ ID NO: 71 and/or SEQ ID NO: 73 in said Fab
while retaining binding specificity for CGRP.
In one embodiment described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab8. In another embodiment described herein, anti-
CGRP antibodies such as Ab8 or Fab fragments thereof may be produced via expression in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab9
In one embodiment, the present disclosure includes chimeric antibodies having
binding specificity to CGRP and possessing a variable light chain sequence comprising the
sequence set forth below:
QVLTQTPSPVSAAVGSTVTINCQASQNVYNNNYLAWYQQKPGQPPKQLIYSTSTL
ASGVSSRFRGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSRGDCFVFGGGTEVV
VKR (SEQ ID NO: 81).
The present disclosure also includes chimeric antibodies having binding
specificity to CGRP and possessing a light chain sequence comprising the sequence set
forth below:
QVLTQTPSPVSAAVGSTVTINCQASQNVYNNNYLAWYQQKPGQPPKQLIYSTSTL
ASGVSSRFRGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSRGDCFVFGGGTEVV
VKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 82).
The present disclosure further includes chimeric antibodies having binding
specificity to CGRP and possessing a variable heavy chain sequence comprising the
sequence set forth below:
QSLEESGGRLVTPGTPLTLTCTVSGIGLSSYYMQWVRQSPGRGLEWIGVIGSDGKT
YYATWAKGRFTISKTSSTTVDLRMASLTTEDTATYFCTRGDIWGPGTLVTVSS
(SEQ ID NO: 83).
The present disclosure also includes chimeric antibodies having binding
specificity to CGRP and possessing a heavy chain sequence comprising the sequence set
forth below:
QSLEESGGRLVTPGTPLTLTCTVSGIGLSSYYMQWVRQSPGRGLEWIGVIGSDGKT
YYATWAKGRFTISKTSSTTVDLRMASLTTEDTATYFCTRGDIWGPGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO: 84).
The present disclosure further contemplates antibodies comprising one or more
of the polypeptide sequences of SEQ ID NO: 85; SEQ ID NO: 86; and SEQ ID NO: 87
which correspond to the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 81 or the light chain sequence
of SEQ ID NO: 82, and/or one or more of the polypeptide sequences of SEQ ID NO: 88;
SEQ ID NO: 89; and SEQ ID NO: 90 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 83 or the heavy chain sequence of SEQ ID NO: 84, or combinations of
these polypeptide sequences. In another embodiment described herein, the antibodies or
fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment described herein, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 81 or SEQ
ID NO: 82. In another embodiment described herein, antibody fragments comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 83 or SEQ ID NO: 84.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 85; SEQ ID NO: 86; and SEQ ID NO: 87 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 81 or the light chain sequence of SEQ
ID NO: 82.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 88; SEQ ID NO: 89; and SEQ ID NO: 90 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable heavy chain sequence of SEQ ID NO: 83 or the heavy chain sequence of
SEQ ID NO: 84.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment described herein,
fragments of the antibodies having binding specificity to CGRP comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 81; the variable heavy chain region of SEQ ID
NO: 83; the complementarity-determining regions (SEQ ID NO: 85; SEQ ID NO: 86; and
SEQ ID NO: 87) of the variable light chain region of SEQ ID NO: 81; and the
complementarity-determining regions (SEQ ID NO: 88; SEQ ID NO: 89; and SEQ ID NO:
90) of the variable heavy chain region of SEQ ID NO: 83.
In a particularly preferred embodiment described herein, the chimeric anti-
CGRP antibody is Ab9, comprising, or alternatively consisting of, SEQ ID NO: 82 and
SEQ ID NO: 84, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment described herein, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab9, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 81 and the variable heavy chain
sequence of SEQ ID NO: 83. This embodiment described herein further contemplates
additions, deletions, and variants of SEQ ID NO: 81 and/or SEQ ID NO: 83 in said Fab
while retaining binding specificity for CGRP.
In one embodiment described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab9. In another embodiment described herein, anti-
CGRP antibodies such as Ab9 or Fab fragments thereof may be produced via expression in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab10
In one embodiment, the present disclosure includes humanized antibodies having
binding specificity to CGRP and possessing a variable light chain sequence comprising the
sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQNVYNNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSRGDCFVFGGGTKVEIK
R (SEQ ID NO: 91).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a light chain sequence comprising the sequence set
forth below:
QVLTQSPSSLSASVGDRVTINCQASQNVYNNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSRGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 92).
The present disclosure further includes humanized antibodies having binding
specificity to CGRP and possessing a variable heavy chain sequence comprising the
sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIGLSSYYMQWVRQAPGKGLEWVGVIGSD
GKTYYATWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCTRGDIWGQGTLVT
VSS (SEQ ID NO: 93).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a heavy chain sequence comprising the sequence set
forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIGLSSYYMQWVRQAPGKGLEWVGVIGSD
GKTYYATWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCTRGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 94).
The present disclosure further contemplates antibodies comprising one or more
of the polypeptide sequences of SEQ ID NO: 95; SEQ ID NO: 96; and SEQ ID NO: 97
which correspond to the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 91 or the light chain sequence
of SEQ ID NO: 92, and/or one or more of the polypeptide sequences of SEQ ID NO: 98;
SEQ ID NO: 99; and SEQ ID NO: 100 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 93 or the heavy chain sequence of SEQ ID NO: 94, or combinations of
these polypeptide sequences. In another embodiment described herein, the antibodies or
fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment described herein, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 91 or SEQ
ID NO: 92. In another embodiment described herein, antibody fragments comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 93 or SEQ ID NO: 94.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 95; SEQ ID NO: 96; and SEQ ID NO: 97 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 91 or the light chain sequence of SEQ
ID NO: 92.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 98; SEQ ID NO: 99; and SEQ ID NO: 100 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable heavy chain sequence of SEQ ID NO: 93 or the heavy chain sequence of
SEQ ID NO: 94.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment described herein,
fragments of the antibodies having binding specificity to CGRP comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 91; the variable heavy chain region of SEQ ID
NO: 93; the complementarity-determining regions (SEQ ID NO: 95; SEQ ID NO: 96; and
SEQ ID NO: 97) of the variable light chain region of SEQ ID NO: 91; and the
complementarity-determining regions (SEQ ID NO: 98; SEQ ID NO: 99; and SEQ ID NO:
100) of the variable heavy chain region of SEQ ID NO: 93.
In a particularly preferred embodiment described herein, the humanized anti-
CGRP antibody is Ab10, comprising, or alternatively consisting of, SEQ ID NO: 92 and
SEQ ID NO: 94, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment described herein, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab10, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 91 and the variable heavy chain
sequence of SEQ ID NO: 93. This embodiment described herein further contemplates
additions, deletions, and variants of SEQ ID NO: 91 and/or SEQ ID NO: 93 in said Fab
while retaining binding specificity for CGRP.
In one embodiment described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab10. In another embodiment described herein, anti-
CGRP antibodies such as Ab10 or Fab fragments thereof may be produced via expression
in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab11
In one embodiment, the present disclosure includes chimeric antibodies having
binding specificity to CGRP and possessing a variable light chain sequence comprising the
sequence set forth below:
QVLTQTASPVSPAVGSTVTINCRASQSVYYNNYLAWYQQKPGQPPKQLIYSTSTLA
SGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSNGDCFVFGGGTEVVV
KR (SEQ ID NO: 101).
The present disclosure also includes chimeric antibodies having binding
specificity to CGRP and possessing a light chain sequence comprising the sequence set
forth below:
QVLTQTASPVSPAVGSTVTINCRASQSVYYNNYLAWYQQKPGQPPKQLIYSTSTLA
SGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSNGDCFVFGGGTEVVV
KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 102).
The present disclosure further includes chimeric antibodies having binding
specificity to CGRP and possessing a variable heavy chain sequence comprising the
sequence set forth below:
QSLEESGGRLVTPGGSLTLTCTVSGIDVTNYYMQWVRQAPGKGLEWIGVIGVNGK
RYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSS
(SEQ ID NO: 103).
The present disclosure also includes chimeric antibodies having binding
specificity to CGRP and possessing a heavy chain sequence comprising the sequence set
forth below:
QSLEESGGRLVTPGGSLTLTCTVSGIDVTNYYMQWVRQAPGKGLEWIGVIGVNGK
RYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO: 104).
The present disclosure further contemplates antibodies comprising one or more
of the polypeptide sequences of SEQ ID NO: 105; SEQ ID NO: 106; and SEQ ID NO: 107
which correspond to the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 101 or the light chain
sequence of SEQ ID NO: 102, and/or one or more of the polypeptide sequences of SEQ ID
NO: 108; SEQ ID NO: 109; and SEQ ID NO: 110 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
heavy chain sequence of SEQ ID NO: 103 or the heavy chain sequence of SEQ ID NO:
104, or combinations of these polypeptide sequences. In another embodiment described
herein, the antibodies or fragments thereof comprise, or alternatively consist of,
combinations of one or more of the CDRs, the variable heavy and variable light chain
sequences, and the heavy and light chain sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment described herein, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 101 or SEQ
ID NO: 102. In another embodiment described herein, antibody fragments comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 103 or SEQ ID NO: 104.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 105; SEQ ID NO: 106; and SEQ ID NO: 107 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 101 or the light chain sequence of SEQ
ID NO: 102.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 108; SEQ ID NO: 109; and SEQ ID NO: 110 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable heavy chain sequence of SEQ ID NO: 103 or the heavy chain sequence of
SEQ ID NO: 104.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment described herein,
fragments of the antibodies having binding specificity to CGRP comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 101; the variable heavy chain region of SEQ ID
NO: 103; the complementarity-determining regions (SEQ ID NO: 105; SEQ ID NO: 106;
and SEQ ID NO: 107) of the variable light chain region of SEQ ID NO: 101; and the
complementarity-determining regions (SEQ ID NO: 108; SEQ ID NO: 109; and SEQ ID
NO: 110) of the variable heavy chain region of SEQ ID NO: 103.
In a particularly preferred embodiment described herein, the chimeric anti-
CGRP antibody is Ab11, comprising, or alternatively consisting of, SEQ ID NO: 102 and
SEQ ID NO: 104, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment described herein, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab11, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 101 and the variable heavy chain
sequence of SEQ ID NO: 103. This embodiment described herein further contemplates
additions, deletions, and variants of SEQ ID NO: 101 and/or SEQ ID NO: 103 in said Fab
while retaining binding specificity for CGRP.
In one embodiment described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab11. In another embodiment described herein, anti-
CGRP antibodies such as Ab11 or Fab fragments thereof may be produced via expression
in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab12
In one embodiment, the present disclosure includes humanized antibodies having
binding specificity to CGRP and possessing a variable light chain sequence comprising the
sequence set forth below:
QVLTQSPSSLSASVGDRVTINCRASQSVYYNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSNGDCFVFGGGTKVEIK
R (SEQ ID NO: 111).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a light chain sequence comprising the sequence set
forth below:
QVLTQSPSSLSASVGDRVTINCRASQSVYYNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSNGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 112).
The present disclosure further includes humanized antibodies having binding
specificity to CGRP and possessing a variable heavy chain sequence comprising the
sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDVTNYYMQWVRQAPGKGLEWVGVIGVN
GKRYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSS (SEQ ID NO: 113).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a heavy chain sequence comprising the sequence set
forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDVTNYYMQWVRQAPGKGLEWVGVIGVN
GKRYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 114).
The present disclosure further contemplates antibodies comprising one or more
of the polypeptide sequences of SEQ ID NO: 115; SEQ ID NO: 116; and SEQ ID NO: 117
which correspond to the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 111 or the light chain
sequence of SEQ ID NO: 112, and/or one or more of the polypeptide sequences of SEQ ID
NO: 118; SEQ ID NO: 119; and SEQ ID NO: 120 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
heavy chain sequence of SEQ ID NO: 113 or the heavy chain sequence of SEQ ID NO:
114, or combinations of these polypeptide sequences. In another embodiment described
herein, the antibodies or fragments thereof comprise, or alternatively consist of,
combinations of one or more of the CDRs, the variable heavy and variable light chain
sequences, and the heavy and light chain sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment described herein, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 111 or SEQ
ID NO: 112. In another embodiment described herein, antibody fragments comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 113 or SEQ ID NO: 114.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 115; SEQ ID NO: 116; and SEQ ID NO: 117 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 111 or the light chain sequence of SEQ
ID NO: 112.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 118; SEQ ID NO: 119; and SEQ ID NO: 120 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable heavy chain sequence of SEQ ID NO: 113 or the heavy chain sequence of
SEQ ID NO: 114.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment described herein,
fragments of the antibodies having binding specificity to CGRP comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 111; the variable heavy chain region of SEQ ID
NO: 113; the complementarity-determining regions (SEQ ID NO: 115; SEQ ID NO: 116;
and SEQ ID NO: 117) of the variable light chain region of SEQ ID NO: 111; and the
complementarity-determining regions (SEQ ID NO: 118; SEQ ID NO: 119; and SEQ ID
NO: 120) of the variable heavy chain region of SEQ ID NO: 113.
In a particularly preferred embodiment described herein, the humanized anti-
CGRP antibody is Ab12, comprising, or alternatively consisting of, SEQ ID NO: 112 and
SEQ ID NO: 114, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment described herein, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab12, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 111 and the variable heavy chain
sequence of SEQ ID NO: 113. This embodiment described herein further contemplates
additions, deletions, and variants of SEQ ID NO: 111 and/or SEQ ID NO: 113 in said Fab
while retaining binding specificity for CGRP.
In one embodiment described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab12. In another embodiment described herein, anti-
CGRP antibodies such as Ab12 or Fab fragments thereof may be produced via expression
in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab13
In one embodiment, the present disclosure includes chimeric antibodies having
binding specificity to CGRP and possessing a variable light chain sequence comprising the
sequence set forth below:
AIVMTQTPSSKSVPVGDTVTINCQASESLYNNNALAWFQQKPGQPPKRLIYDASKL
ASGVPSRFSGGGSGTQFTLTISGVQCDDAATYYCGGYRSDSVDGVAFAGGTEVVV
KR (SEQ ID NO: 121).
The present disclosure also includes chimeric antibodies having binding
specificity to CGRP and possessing a light chain sequence comprising the sequence set
forth below:
AIVMTQTPSSKSVPVGDTVTINCQASESLYNNNALAWFQQKPGQPPKRLIYDASKL
ASGVPSRFSGGGSGTQFTLTISGVQCDDAATYYCGGYRSDSVDGVAFAGGTEVVV
KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 122).
The present disclosure further includes chimeric antibodies having binding
specificity to CGRP and possessing a variable heavy chain sequence comprising the
sequence set forth below:
QSVEESGGGLVQPEGSLTLTCTASGFDFSSNAMWWVRQAPGKGLEWIGIIYNGDG
STYYASWVNGRFSISKTSSTTVTLQLNSLTVADTATYYCARDLDLWGPGTLVTVS
S (SEQ ID NO: 123).
The present disclosure also includes chimeric antibodies having binding
specificity to CGRP and possessing a heavy chain sequence comprising the sequence set
forth below:
QSVEESGGGLVQPEGSLTLTCTASGFDFSSNAMWWVRQAPGKGLEWIGCIYNGD
GSTYYASWVNGRFSISKTSSTTVTLQLNSLTVADTATYYCARDLDLWGPGTLVTV
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 124).
The present disclosure further contemplates antibodies comprising one or more
of the polypeptide sequences of SEQ ID NO: 125; SEQ ID NO: 126; and SEQ ID NO: 127
which correspond to the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 121 or the light chain
sequence of SEQ ID NO: 122, and/or one or more of the polypeptide sequences of SEQ ID
NO: 128; SEQ ID NO: 129; and SEQ ID NO: 130 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
heavy chain sequence of SEQ ID NO: 123 or the heavy chain sequence of SEQ ID NO:
124, or combinations of these polypeptide sequences. In another embodiment described
herein, the antibodies or fragments thereof comprise, or alternatively consist of,
combinations of one or more of the CDRs, the variable heavy and variable light chain
sequences, and the heavy and light chain sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment described herein, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 121 or SEQ
ID NO: 122. In another embodiment described herein, antibody fragments comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 123 or SEQ ID NO: 124.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 125; SEQ ID NO: 126; and SEQ ID NO: 127 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 121 or the light chain sequence of SEQ
ID NO: 122.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 128; SEQ ID NO: 129; and SEQ ID NO: 130 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable heavy chain sequence of SEQ ID NO: 123 or the heavy chain sequence of
SEQ ID NO: 124.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment described herein,
fragments of the antibodies having binding specificity to CGRP comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 121; the variable heavy chain region of SEQ ID
NO: 123; the complementarity-determining regions (SEQ ID NO: 125; SEQ ID NO: 126;
and SEQ ID NO: 127) of the variable light chain region of SEQ ID NO: 121; and the
complementarity-determining regions (SEQ ID NO: 128; SEQ ID NO: 129; and SEQ ID
NO: 130) of the variable heavy chain region of SEQ ID NO: 123.
In a particularly preferred embodiment described herein, the chimeric anti-
CGRP antibody is Ab13, comprising, or alternatively consisting of, SEQ ID NO: 122 and
SEQ ID NO: 124, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment described herein, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab13, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 121 and the variable heavy chain
sequence of SEQ ID NO: 123. This embodiment described herein further contemplates
additions, deletions, and variants of SEQ ID NO: 121 and/or SEQ ID NO: 123 in said Fab
while retaining binding specificity for CGRP.
In one embodiment described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab13. In another embodiment described herein, anti-
CGRP antibodies such as Ab13 or Fab fragments thereof may be produced via expression
in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab14
In one embodiment, the present disclosure includes humanized antibodies having
binding specificity to CGRP and possessing a variable light chain sequence comprising the
sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQNVYNNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSRGDCFVFGGGTKVEIK
R (SEQ ID NO: 131).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a light chain sequence comprising the sequence set
forth below:
QVLTQSPSSLSASVGDRVTINCQASQNVYNNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSRGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 132).
The present disclosure further includes humanized antibodies having binding
specificity to CGRP and possessing a variable heavy chain sequence comprising the
sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIGLSSYYMQWVRQAPGKGLEWVGVIGSD
GKTYYATWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCTRGDIWGQGTLVT
VSS (SEQ ID NO: 133).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP and possessing a heavy chain sequence comprising the sequence set
forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIGLSSYYMQWVRQAPGKGLEWVGVIGSD
GKTYYATWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCTRGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 134).
The present disclosure further contemplates antibodies comprising one or more
of the polypeptide sequences of SEQ ID NO: 135; SEQ ID NO: 136; and SEQ ID NO: 137
which correspond to the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 131 or the light chain
sequence of SEQ ID NO: 132, and/or one or more of the polypeptide sequences of SEQ ID
NO: 138; SEQ ID NO: 139; and SEQ ID NO: 140 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
heavy chain sequence of SEQ ID NO: 133 or the heavy chain sequence of SEQ ID NO:
134, or combinations of these polypeptide sequences. In another embodiment described
herein, the antibodies or fragments thereof comprise, or alternatively consist of,
combinations of one or more of the CDRs, the variable heavy and variable light chain
sequences, and the heavy and light chain sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment described herein, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 131 or SEQ
ID NO: 132. In another embodiment described herein, antibody fragments comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 133 or SEQ ID NO: 134.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 135; SEQ ID NO: 136; and SEQ ID NO: 137 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 131 or the light chain sequence of SEQ
ID NO: 132.
In a further embodiment described herein, fragments of the antibody having
binding specificity to CGRP comprise, or alternatively consist of, one or more of the
polypeptide sequences of SEQ ID NO: 138; SEQ ID NO: 139; and SEQ ID NO: 140 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable heavy chain sequence of SEQ ID NO: 133 or the heavy chain sequence of
SEQ ID NO: 134.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment described herein,
fragments of the antibodies having binding specificity to CGRP comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 131; the variable heavy chain region of SEQ ID
NO: 133; the complementarity-determining regions (SEQ ID NO: 135; SEQ ID NO: 136;
and SEQ ID NO: 137) of the variable light chain region of SEQ ID NO: 131; and the
complementarity-determining regions (SEQ ID NO: 138; SEQ ID NO: 139; and SEQ ID
NO: 140) of the variable heavy chain region of SEQ ID NO: 133.
In a particularly preferred embodiment described herein, the humanized anti-
CGRP antibody is Ab14, comprising, or alternatively consisting of, SEQ ID NO: 132 and
SEQ ID NO: 134, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment described herein, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab14, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 131 and the variable heavy chain
sequence of SEQ ID NO: 133. This embodiment described herein further contemplates
additions, deletions, and variants of SEQ ID NO: 131 and/or SEQ ID NO: 133 in said Fab
while retaining binding specificity for CGRP.
In one embodiment described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab14. In another embodiment described herein, anti-
CGRP antibodies such as Ab14 or Fab fragments thereof may be produced via expression
in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
In another embodiment, antibody fragments may be present in one or more of
the following non-limiting forms: Fab, Fab', F(ab') , Fv and single chain Fv antibody
forms. In a preferred embodiment, the anti-CGRP antibodies described herein further
comprises the kappa constant light chain sequence comprising the sequence set forth
below:
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 283).
In another preferred embodiment, the anti-CGRP antibodies described herein
further comprises the gamma-1 constant heavy chain polypeptide sequence comprising the
sequence set forth below:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGK (SEQ ID NO: 284).
In another embodiment, the present disclosure contemplates an isolated anti-
CGRP antibody comprising a V polypeptide sequence selected from: SEQ ID NO: 3, 13,
23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, or 133, or a variant thereof; and further
comprising a V polypeptide sequence selected from: SEQ ID NO: 1, 11, 21, 31, 41, 51,
61, 71, 81, 91, 101, 111, 121, or 131, or a variant thereof, wherein one or more of the
framework residues (FR residues) in said V V polypeptide has been substituted with
H or L
another amino acid residue resulting in an anti-CGRP antibody that specifically binds
CGRP. The present disclosure contemplates humanized and chimeric forms of these
antibodies. The chimeric antibodies may include an Fc derived from IgG1, IgG2, IgG3,
IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16,
IgG17, IgG18 or IgG19 constant regions.
In one embodiment described herein, the antibodies or V or V polypeptides
originate or are selected from one or more rabbit B cell populations prior to initiation of the
humanization process referenced herein.
In another embodiment described herein, the anti-CGRP antibodies and
fragments thereof do not have binding specificity for CGRP-R. In a further embodiment
described herein, the anti-CGRP antibodies and fragments thereof inhibit the association of
CGRP with CGRP-R. In another embodiment described herein, the anti-CGRP antibodies
and fragments thereof inhibit the association of CGRP with CGRP-R and/or additional
proteins and/or multimers thereof, and/or antagonizes the biological effects thereof.
As stated in paragraph [0127] herein, antibodies and fragments thereof may be
modified post-translationally to add effector moieties such as chemical linkers, detectable
moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent
materials, radioactive materials, and chemiluminescent moieties, or functional moieties
such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and
radioactive materials.
Antibodies or fragments thereof may also be chemically modified to provide
additional advantages such as increased solubility, stability and circulating time (in vivo
half-life) of the polypeptide, or decreased immunogenicity (See U.S. Pat. No. 4,179,337).
The chemical moieties for derivatization may be selected from water soluble polymers such
as polyethylene glycol, ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The antibodies and
fragments thereof may be modified at random positions within the molecule, or at
predetermined positions within the molecule and may include one, two, three or more
attached chemical moieties.
The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between about 1
kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene
glycol, some molecules will weigh more, some less, than the stated molecular weight) for
ease in handling and manufacturing. Other sizes may be used, depending on the desired
therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on
biological activity, the ease in handling, the degree or lack of antigenicity and other known
effects of the polyethylene glycol to a therapeutic protein or analog). For example, the
polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500,
2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500,
9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000,
14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500,
,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000,
80,000, 85,000, 90,000, 95,000, or 100,000 kDa. Branched polyethylene glycols are
described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem.
Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750
(1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of
which are incorporated herein by reference.
There are a number of attachment methods available to those skilled in the art,
See e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), See
also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF
using tresyl chloride). For example, polyethylene glycol may be covalently bound through
amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive
groups are those to which an activated polyethylene glycol molecule may be bound. The
amino acid residues having a free amino group may include lysine residues and the N-
terminal amino acid residues; those having a free carboxyl group may include aspartic acid
residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups
may also be used as a reactive group for attaching the polyethylene glycol molecules.
Preferred for therapeutic purposes is attachment at an amino group, such as attachment at
the N-terminus or lysine group.
As suggested above, polyethylene glycol may be attached to proteins via linkage
to any of a number of amino acid residues. For example, polyethylene glycol can be linked
to polypeptides via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or
cysteine residues. One or more reaction chemistries may be employed to attach
polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid,
glutamic acid, or cysteine) or to more than one type of amino acid residue (e.g., lysine,
histidine, aspartic acid, glutamic acid, cysteine and combinations thereof).
Alternatively, antibodies or fragments thereof may have increased in vivo half
lives via fusion with albumin (including but not limited to recombinant human serum
albumin or fragments or variants thereof (See, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2,
1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein
incorporated by reference in their entirety)) or other circulating blood proteins such as
transferrin or ferritin. In a preferred embodiment, polypeptides and/or antibodies of the
present present disclosure (including fragments or variants thereof) are fused with the
mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as
shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference
in its entirety. Polynucleotides encoding fusion proteins described herein are also
encompassed by the present disclosure.
Regarding detectable moieties, further exemplary enzymes include, but are not
limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-
galactosidase and luciferase. Further exemplary fluorescent materials include, but are not
limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone,
dichlorotriazinylamine, phycoerythrin and dansyl chloride. Further exemplary
chemiluminescent moieties include, but are not limited to, luminol. Further exemplary
bioluminescent materials include, but are not limited to, luciferin and aequorin. Further
exemplary radioactive materials include, but are not limited to, Iodine 125 ( I), Carbon 14
14 35 3 32
( C), Sulfur 35 ( S), Tritium ( H) and Phosphorus 32 ( P).
Regarding functional moieties, exemplary cytotoxic agents include, but are not
limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-
fluorouracil decarbazine; alkylating agents such as mechlorethamine, thioepa chlorambucil,
melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea,
cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin,
mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin
(paraplatin); anthracyclines include daunorubicin (formerly daunomycin), doxorubicin
(adriamycin), detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone and
bisantrene; antibiotics include dactinomycin (actinomycin D), bleomycin, calicheamicin,
mithramycin, and anthramycin (AMC); and antimytotic agents such as the vinca alkaloids,
vincristine and vinblastine. Other cytotoxic agents include paclitaxel (taxol), ricin,
pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide,
emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol,
puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane (O,P'-
(DDD)), interferons, and mixtures of these cytotoxic agents.
Further cytotoxic agents include, but are not limited to, chemotherapeutic agents
such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-
fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine and bleomycin.
Toxic enzymes from plants and bacteria such as ricin, diphtheria toxin and Pseudomonas
toxin may be conjugated to the humanized or chimeric antibodies, or binding fragments
thereof, to generate cell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad. Sci.
USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick,
et al., Proc. Nat'l Acad. Sci. USA 77:5419 (1980)).
Other cytotoxic agents include cytotoxic ribonucleases as described by
Goldenberg in U.S. Pat. No. 6,653,104. Embodiments described herein also relate to
radioimmunoconjugates where a radionuclide that emits alpha or beta particles is stably
coupled to the antibody, or binding fragments thereof, with or without the use of a
complex-forming agent. Such radionuclides include beta-emitters such as Phosphorus-32
32 47 67 67 88
( P), Scandium-47 ( Sc), Copper-67 ( Cu), Gallium-67 ( Ga), Yttrium-88 ( Y),
90 125 131 153
Yttrium-90 ( Y), Iodine-125 ( I), Iodine-131 ( I), Samarium-153 ( Sm), Lutetium-
177 186 188
177 ( Lu), Rhenium-186 ( Re) or Rhenium-188 ( Re), and alpha-emitters such as
211 212 212 213
Astatine-211 ( At), Lead-212 ( Pb), Bismuth-212 ( Bi) or -213 ( Bi) or Actinium-
225 ( Ac).
Methods are known in the art for conjugating an antibody or binding fragment
thereof to a detectable moiety and the like, such as for example those methods described by
Hunter et al, Nature 144:945 (1962); David et al, Biochemistry 13:1014 (1974); Pain et al,
J. Immunol. Meth. 40:219 (1981); and Nygren, J., Histochem. and Cytochem. 30:407
(1982).
Embodiments described herein further include variants and equivalents that are
substantially homologous to the antibodies, antibody fragments, diabodies, SMIPs,
camelbodies, nanobodies, IgNAR, polypeptides, variable regions and CDRs set forth
herein. These may contain, e.g., conservative substitution mutations, (i.e., the substitution
of one or more amino acids by similar amino acids). For example, conservative
substitution refers to the substitution of an amino acid with another within the same general
class, e.g., one acidic amino acid with another acidic amino acid, one basic amino acid with
another basic amino acid, or one neutral amino acid by another neutral amino acid. What is
intended by a conservative amino acid substitution is well known in the art.
In another embodiment, the present disclosure contemplates polypeptide
sequences having at least 90% or greater sequence homology to any one or more of the
polypeptide sequences of antibody fragments, variable regions and CDRs set forth herein.
More preferably, the present disclosure contemplates polypeptide sequences having at least
95% or greater sequence homology, even more preferably at least 98% or greater sequence
homology, and still more preferably at least 99% or greater sequence homology to any one
or more of the polypeptide sequences of antibody fragments, variable regions and CDRs set
forth herein. Methods for determining homology between nucleic acid and amino acid
sequences are well known to those of ordinary skill in the art.
In another embodiment, the present disclosure further contemplates the above-
recited polypeptide homologs of the antibody fragments, variable regions and CDRs set
forth herein further having anti-CGRP activity. Non-limiting examples of anti-CGRP
activity are set forth herein, for example, in paragraphs [0329]-[0350] infra.
In another embodiment, the present disclosure further contemplates the
generation and use of anti-idiotypic antibodies that bind any of the foregoing sequences. In
an exemplary embodiment, such an anti-idiotypic antibody could be administered to a
subject who has received an anti-CGRP antibody to modulate, reduce, or neutralize, the
effect of the anti-CGRP antibody. Such anti-idiotypic antibodies could also be useful for
treatment of an autoimmune disease characterized by the presence of anti-CGRP
antibodies. A further exemplary use of such anti-idiotypic antibodies is for detection of the
anti-CGRP antibodies described herein, for example to monitor the levels of the anti-CGRP
antibodies present in a subject’s blood or other bodily fluids.
The present present disclosure also contemplates anti-CGRP antibodies
comprising any of the polypeptide or polynucleotide sequences described herein substituted
for any of the other polynucleotide sequences described herein. For example, without
limitation thereto, the present dislcosure contemplates antibodies comprising the
combination of any of the variable light chain and variable heavy chain sequences
described herein, and further contemplates antibodies resulting from substitution of any of
the CDR sequences described herein for any of the other CDR sequences described herein.
Additional Exemplary Embodiments of the Invention
In another embodiment, the present disclosure contemplates one or more anti-
human CGRP antibodies or antibody fragments thereof which specifically bind to the same
ovelappping linear or conformational epitope(s) and/or competes for binding to the same
ovelappping linear or conformational epitope(s) on an intact human CGRP polypeptide or
fragment thereof as an anti-human CGRP antibody selected from Ab1, Ab2, Ab3, Ab4,
Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, or Ab14. In a preferred
embodiment, the anti-human CGRP antibody or fragment thereof specifically binds to the
same ovelappping linear or conformational epitope(s) and/or competes for binding to the
same ovelappping linear or conformational epitope(s) on an intact human CGRP
polypeptide or a fragment thereof as Ab3, Ab6, Ab13, or Ab14.
A preferred embodiment described herein is directed to chimeric or humanized
antibodies and fragments thereof (including Fab fragments) having binding specificity for
CGRP and inhibiting biological activities mediated by the binding of CGRP to the CGRP
receptor. In a particularly preferred embodiment described herein, the chimeric or
humanized anti-CGRP antibodies are selected from Ab3, Ab6, Ab13, or Ab14.
In a further embodiment described herein is contemplated a method of reducing,
treating or preventing diseases or disorders associated with CGRP by affecting those
biological activities mediated via CGRP, thereby avoiding the biological activities
mediated via binding of CGRP to CGRP-R. In one embodiment, the disease or disorder
associated with CGRP is migraine or another disorder wherein CGRP elicits pain,
headache, pain, cancer, overactive bladder, or weightloss. A further non-limiting listing of
diseases and disorders associated with CGRP is provided herein.
Another preferred embodiment described herein contemplates the use of Fab
polypeptide sequences for the treatment of migraines and headaches in a patient. Non-
limiting types of migraines and headaches that may be treated using Fab polypeptide
sequences are provided elsewhere in this disclosure.
In another embodiment described herein, the anti-human CGRP antibody is an
antibody which specifically binds to the same ovelappping linear or conformational
epitopes on an intact CGRP polypeptide or fragment thereof that is (are) specifically bound
by Ab3, Ab6, Ab13, or Ab14 as ascertained by epitopic mapping using overlapping linear
peptide fragments which span the full length of the native human CGRP polypeptide.
The present disclosure is also directed to an anti-CGRP antibody that binds with
the same CGRP epitope and/or competes with an anti-CGRP antibody for binding to CGRP
as an antibody or antibody fragment disclosed herein, including but not limited to an anti-
CGRP antibody selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10,
Ab11, Ab12, Ab13, or Ab14.
In another embodiment, the present disclosure is also directed to an isolated anti-
CGRP antibody or antibody fragment comprising one or more of the CDRs contained in
the VH polypeptide sequences selected from: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113,
123, or 133, or a variant thereof, and/or one or more of the CDRs contained in the V
polypeptide sequences selected from: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, or
131, or a variant thereof.
In one embodiment described herein, the anti-human CGRP antibody discussed
in the two prior paragraphs comprises at least 2 complementarity determining regions
(CDRs) in each the variable light and the variable heavy regions which are identical to
those contained in an anti-human CGRP antibody selected from Ab1, Ab2, Ab3, Ab4, Ab5,
Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, or Ab14.
In a preferred embodiment, the anti-human CGRP antibody discussed above
comprises at least 2 complementarity determining regions (CDRs) in each the variable light
and the variable heavy regions which are identical to those contained in Ab3 or Ab6. In
another embodiment, all of the CDRs of the anti-human CGRP antibody discussed above
are identical to the CDRs contained in an anti-human CGRP antibody selected from Ab1,
Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, or Ab14. In a
preferred embodiment described herein, all of the CDRs of the anti-human CGRP antibody
discussed above are identical to the CDRs contained in an anti-human CGRP antibody
selected from Ab3 or Ab6.
The present disclosure further contemplates that the one or more anti-human
CGRP antibodies discussed above are aglycosylated; that contain an Fc region that has
been modified to alter effector function, half-life, proteolysis, and/or glycosylation; are
human, humanized, single chain or chimeric; and are a humanized antibody derived from a
rabbit (parent) anti-human CGRP antibody.
The present disclosure further contemplates one or more anti-human CGRP
antibodies wherein the framework regions (FRs) in the variable light region and the
variable heavy regions of said antibody respectively are human FRs which are unmodified
or which have been modified by the substitution of one or more human FR residues in the
variable light or heavy chain region with the corresponding FR residues of the parent rabbit
antibody, and wherein said human FRs have been derived from human variable heavy and
light chain antibody sequences which have been selected from a library of human germline
antibody sequences based on their high level of homology to the corresponding rabbit
variable heavy or light chain regions relative to other human germline antibody sequences
contained in the library.
In one embodiment described herein, the anti-human CGRP antibody or
fragment specifically binds to CGRP expressing human cells and/or to circulating soluble
CGRP molecules in vivo, including CGRP expressed on or by human cells in a patient with
a disease associated with cells that express CGRP.
In another embodiment, the disease is selected from migraines (with or without
aura), weight loss, cancer or tumors, angiogenesis associated with cancer or tumor growth,
angiogenesis associated with cancer or tumor survival, hemiplagic migraines, cluster
headaches, migrainous neuralgia, chronic headaches, tension headaches, general
headaches, hot flushes, chronic paroxysomal hemicrania, secondary headaches due to an
underlying structural problem in the head or neck, cranial neuralgia, sinus headaches (such
as for example associated with sinusitis), allergy-induced headaches or migraines, pain,
inflammatory pain, post-operative incision pain, complex regional pain syndrome, cancer
pain, primary or metastatic bone cancer pain, fracture pain, chronic pain, osteoporotic
fracture pain, pain resulting from burn, osteoporosis, gout joint pain, abdominal pain, pain
associated with sickle cell crises, and other nociceptic pain, as well as hepatocellular
carcinoma, breast cancer, liver cirrhosis, neurogenic pain, neuropathic pain, nociceptic
pain, trigeminal neuralgia, post-herpetic neuralgia, phantom limb pain, fibromyalgia,
menstrual pain, ovarialgia, reflex sympathetic dystrophy, neurogenic pain, osteoarthritis or
rheumatoid arthritis pain, lower back pain, diabetic neuropathy, sciatica, or pain or visceral
pain associated with: gastro-esophageal reflux, dyspepsia, irritable bowel syndrome,
irritable colon, spastic colon, mucous colitis, inflammatory bowel disease, Crohn’s disease,
ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstrual period, labor,
menopause, prostatitis, pancreatitis, renal colic, dysmenorrhea, cystitis, including
interstitial cystitis (IC), surgery associated with the ileus, diverticulitis, peritonitis,
pericarditis, hepatitis, appendicitis, colitis, cholecystitis, endometriosis, chronic and/or
acute pancreatitis, myocardial infarction, kidney pain, pleural pain, prostatitis, pelvic pain,
trauma to an organ, chronic nociceptive pain, chronic neuropathic pain, chronic
inflammatory pain, fibromyalgia, breakthrough pain and persistent pain.
In another embodiment described herein, the disease is cancer pain arising from
malignancy or from cancer preferably selected from one or more of: adenocarcinoma in
glandular tissue, blastoma in embryonic tissue of organs, carcinoma in epithelial tissue,
leukemia in tissues that form blood cells, lymphoma in lymphatic tissue, myeloma in bone
marrow, sarcoma in connective or supportive tissue, adrenal cancer, AIDS-related
lymphoma, anemia, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid
tumours, cervical cancer, chemotherapy, colon cancer, cytopenia, endometrial cancer,
esophageal cancer, gastric cancer, head cancer, neck cancer, hepatobiliary cancer, kidney
cancer, leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's disease, lymphoma, non-
Hodgkin's, nervous system tumours, oral cancer, ovarian cancer, pancreatic cancer, prostate
cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer,
urethral cancer, bone cancer, sarcomas cancer of the connective tissue, cancer of bone
tissue, cancer of blood-forming cells, cancer of bone marrow, multiple myeloma,
leukaemia, primary or secondary bone cancer, tumours that metastasize to the bone,
tumours infiltrating the nerve and hollow viscus, tumours near neural structures. Further
preferably the cancer pain comprises visceral pain, preferably visceral pain which arises
from pancreatic cancer and/or metastases in the abdomen. Further preferably the cancer
pain comprises somatic pain, preferably somatic pain due to one or more of bone cancer,
metastasis in the bone, postsurgical pain, sarcomas cancer of the connective tissue, cancer
of bone tissue, cancer of blood-forming cells of the bone marrow, multiple myeloma,
leukaemia, primary or secondary bone cancer.
The present disclosure further contemplates anti-human CGRP antibodies or
fragments directly or indirectly attached to a detectable label or therapeutic agent.
The present disclosure also contemplates one or more nucleic acid sequences
which result in the expression of an anti-human CGRP antibody or antibody fragment as
set forth above, including those comprising, or alternatively consisting of, yeast or human
preferred codons. The present disclosure also contemplates vectors (including plasmids or
recombinant viral vectors) comprising said nucleic acid sequence(s). The present
disclosure also contemplates host cells or recombinant host cells expressing at least one of
the antibodies set forth above, including a mammalian, yeast, bacterial, and insect cells. In
a preferred embodiment, the host cell is a yeast cell. In a further preferred embodiment, the
yeast cell is a diploidal yeast cell. In a more preferred embodiment, the yeast cell is a
Pichia yeast.
The present disclosure also contemplates a method of treatment comprising
administering to a patient with a disease or condition associated with CGRP expressing
cells a therapeutically effective amount of at least one anti-human CGRP antibody or
fragment described herein. The present disclosure also contemplates that the treatment
method may involve the administration of two or more anti-CGRP antibodies or fragments
thereof and disclosed herein. If more than one antibody is administered to the patient, the
multiple antibodies may be administered simultaneously or concurrently, or may be
staggered in their administration. The diseases that may be treated are presented in the
non-limiting list set forth above and elsewhere herein. In a preferred embodiment, the
disease is selected from migraine, headache, weight loss, pain, cancer pain or neuropathic
pain. In another embodiment the treatment further includes the administration of another
therapeutic agent or regimen selected from chemotherapy, radiotherapy, cytokine
administration or gene therapy.
In a non-limiting embodiment described herein, another therapeutic agent or
regimen includes Taxol (paclitaxel) or its derivatives, platinum compounds such as
carboplatin or cisplatin, anthrocyclines such as doxorubicin, alkylating agents such as
cyclophosphamide, anti-metabolites such as 5-fluorouracil, or etoposide.
The present disclosure further contemplates a method of in vivo imaging which
detects the presence of cells which express CGRP comprising administering a
diagnostically effective amount of at least one anti-human CGRP antibody. In one
embodiment, said administration further includes the administration of a radionuclide or
fluorophore that facilitates detection of the antibody at CGRP expressing disease sites. In a
further embodiment, the results of said in vivo imaging method are used to facilitate the
design of an appropriate therapeutic regimen, including therapeutic regimens including
radiotherapy, chemotherapy or a combination thereof.
The anti-CGRP activity of the anti-CGRP antibodies of the present present
disclosure, and fragments thereof having binding specificity to CGRP, may also be
described by their strength of binding or their affinity for CGRP. In one embodiment
described herein, the anti-CGRP antibodies described herein, and fragments thereof having
binding specificity to CGRP, bind to CGRP with a dissociation constant (K ) of less than
-7 -7 -8 -8 -9 -9 -10 -10
or equal to 5x10 M, 10 M, 5x10 M, 10 M, 5x10 M, 10 M, 5x10 M, 10 M,
-11 -11 -12 -12 -13 -13
5x10 M, 10 M, 5x10 M, 10 M, 5x10 M, or 10 M. Preferably, the anti-CGRP
antibodies and fragments thereof bind CGRP with a dissociation constant of less than or
-11 -12 -12
equal to 10 M, 5x10 M, or 10 M. In another embodiment described herein, the anti-
CGRP antibodies described herein, and fragments thereof having binding specificity to
CGRP, bind to a linear or conformational CGRP epitope.
In another embodiment described herein, the anti-CGRP activity of the anti-
CGRP antibodies described herein, and fragments thereof having binding specificity to
-4 -1 -5 -1 -5 -1
CGRP, bind to CGRP with an off-rate of less than or equal to 10 S , 5x10 S , 10 S ,
-6 -1 -6 -1 -7 -1 -7 -1
5x10 S , 10 S , 5x10 S , or 10 S .
In a further embodiment described herein, the anti-CGRP activity of the anti-
CGRP antibodies described herein, and fragments thereof having binding specificity to
CGRP, exhibit anti-CGRP activity by preventing, ameliorating or reducing the symptoms
of, or alternatively treating, diseases and disorders associated with CGRP. Non-limiting
examples of diseases and disorders associated with CGRP are set forth herein.
Polynucleotides Encoding Anti-CGRP Antibody Polypeptides
Antibody Ab1
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment described herein,
polynucleotides comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 1:
CAAGTGCTGACCCAGACTGCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATGATAACAACTACC
TAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCT
ACATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGG
GACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTA
CTACTGTCTAGGCAGTTATGATTGTAGTAGTGGTGATTGTTTTGTTTTCGGCGG
AGGGACCGAGGTGGTGGTCAAACGT (SEQ ID NO: 141).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the light chain polypeptide
sequence of SEQ ID NO: 2:
CAAGTGCTGACCCAGACTGCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATGATAACAACTACC
TAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCT
ACATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGG
GACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTA
CTACTGTCTAGGCAGTTATGATTGTAGTAGTGGTGATTGTTTTGTTTTCGGCGG
AGGGACCGAGGTGGTGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCT
TCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGC
TGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC
CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACA
GCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAA
ACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCA
CAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 142).
In another embodiment described herein, polynucleotides comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 3:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGTCTCTGGACTCGACCTCAGTAGCTACTACATGCA
ATGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTGGTA
TTAATGATAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCC
AGAGCCTCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGA
CACGGCCACCTATTTCTGTGCCAGAGGGGACATCTGGGGCCCAGGCACCCTCG
TCACCGTCTCGAGC (SEQ ID NO: 143).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the heavy chain polypeptide
sequence of SEQ ID NO: 4:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGAC
ACTCACCTGCACAGTCTCTGGACTCGACCTCAGTAGCTACTACATGCAATGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTGGTATTAATG
ATAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGAGCC
TCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGC
CACCTATTTCTGTGCCAGAGGGGACATCTGGGGCCCAGGCACCCTCGTCACCG
TCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCA
AGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA
CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGA
CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT
CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG
TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG
GGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC
ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC
CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG
AACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC
AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA
CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID
NO: 144).
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 145; SEQ ID NO: 146; and SEQ ID
NO: 147 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID
NO: 1 or the light chain sequence of SEQ ID NO: 2.
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 148; SEQ ID NO: 149; and SEQ ID
NO: 150 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID
NO: 3 or the heavy chain sequence of SEQ ID NO: 4.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment described herein, polynucleotides encoding antibody fragments
having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or
more, including all of the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 141 encoding the light chain variable sequence of SEQ ID
NO: 1; the polynucleotide SEQ ID NO: 142 encoding the light chain sequence of SEQ ID
NO: 2; the polynucleotide SEQ ID NO: 143 encoding the heavy chain variable sequence of
SEQ ID NO: 3; the polynucleotide SEQ ID NO: 144 encoding the heavy chain sequence of
SEQ ID NO: 4; polynucleotides encoding the complementarity-determining regions (SEQ
ID NO: 145; SEQ ID NO: 146; and SEQ ID NO: 147) of the light chain variable sequence
of SEQ ID NO: 1 or the light chain sequence of SEQ ID NO: 2; and polynucleotides
encoding the complementarity-determining regions (SEQ ID NO: 148; SEQ ID NO: 149;
and SEQ ID NO: 150) of the heavy chain variable sequence of SEQ ID NO: 3 or the heavy
chain sequence of SEQ ID NO: 4.
In a preferred embodiment described herein, polynucleotides comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for CGRP. With respect to antibody Ab1, the
polynucleotides encoding the full length Ab1 antibody comprise, or alternatively consist of,
the polynucleotide SEQ ID NO: 142 encoding the light chain sequence of SEQ ID NO: 2
and the polynucleotide SEQ ID NO: 144 encoding the heavy chain sequence of SEQ ID
NO: 4.
Another embodiment described herein contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO,
NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as the
yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab1 following expression of the full-length
polynucleotides in a suitable host. In another embodiment described herein, anti-CGRP
antibodies such as Ab1 or Fab fragments thereof may be produced via expression of Ab1
polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect,
or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia)
and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia
pastoris.
Antibody Ab2
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment described herein,
polynucleotides comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 11:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATGATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTAGGCAGTTATGATTGTAGTAGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 151).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the light chain polypeptide
sequence of SEQ ID NO: 12:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATGATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTAGGCAGTTATGATTGTAGTAGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 152).
In another embodiment described herein, polynucleotides comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 13:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGACTCGACCTCAGTAGCTACTACAT
GCAATGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTG
GTATCAATGATAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 153).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the heavy chain polypeptide
sequence of SEQ ID NO: 14:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGACTCGACCTCAGTAGCTACTACATGCAATG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTGGTATCA
ATGATAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATC
TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG
GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCG
TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 154).
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 155; SEQ ID NO: 156; and SEQ ID
NO: 157 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID
NO: 11 or the light chain sequence of SEQ ID NO: 12.
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 158; SEQ ID NO: 159; and SEQ ID
NO: 160 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID
NO: 13 or the heavy chain sequence of SEQ ID NO: 14.
The present disclosurealso contemplates polynucleotide sequences including one
or more of the polynucleotide sequences encoding antibody fragments described herein. In
one embodiment described herein, polynucleotides encoding antibody fragments having
binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 151 encoding the light chain variable sequence of SEQ ID
NO: 11; the polynucleotide SEQ ID NO: 152 encoding the light chain sequence of SEQ ID
NO: 12; the polynucleotide SEQ ID NO: 153 encoding the heavy chain variable sequence
of SEQ ID NO: 13; the polynucleotide SEQ ID NO: 154 encoding the heavy chain
sequence of SEQ ID NO: 14; polynucleotides encoding the complementarity-determining
regions (SEQ ID NO: 155; SEQ ID NO: 156; and SEQ ID NO: 157) of the light chain
variable sequence of SEQ ID NO: 11 or the light chain sequence of SEQ ID NO: 12; and
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 158;
SEQ ID NO: 159; and SEQ ID NO: 160) of the heavy chain variable sequence of SEQ ID
NO: 13 or the heavy chain sequence of SEQ ID NO: 14.
In a preferred embodiment described herein, polynucleotides comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for CGRP. With respect to antibody Ab2, the
polynucleotides encoding the full length Ab2 antibody comprise, or alternatively consist of,
the polynucleotide SEQ ID NO: 152 encoding the light chain sequence of SEQ ID NO: 12
and the polynucleotide SEQ ID NO: 154 encoding the heavy chain sequence of SEQ ID
NO: 14.
Another embodiment described herein contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO,
NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as the
yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab2 following expression of the full-length
polynucleotides in a suitable host. In another embodiment described herein, anti-CGRP
antibodies such as Ab2 or Fab fragments thereof may be produced via expression of Ab2
polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect,
or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia)
and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia
pastoris.
Antibody Ab3
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment described herein,
polynucleotides comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 21:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATGATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTAGGCAGTTATGATTGTAGTAGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 161).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the light chain polypeptide
sequence of SEQ ID NO: 22:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATGATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTAGGCAGTTATGATTGTAGTAGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 162).
In another embodiment described herein, polynucleotides comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 23:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGACTCGACCTCAGTAGCTACTACAT
GCAATGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTG
GTATCAATGATAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 163).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the heavy chain polypeptide
sequence of SEQ ID NO: 24:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGACTCGACCTCAGTAGCTACTACATGCAATG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTGGTATCA
ATGATAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACGCGAGAGTTGAGCCCAAATCT
TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG
ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG
GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG
CCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGT
CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 164).
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 165; SEQ ID NO: 166; and SEQ ID
NO: 167 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID
NO: 21 or the light chain sequence of SEQ ID NO: 22.
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 168; SEQ ID NO: 169; and SEQ ID
NO: 170 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID
NO: 23 or the heavy chain sequence of SEQ ID NO: 24.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment described herein, polynucleotides encoding antibody fragments
having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or
more, including all of the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 161 encoding the light chain variable sequence of SEQ ID
NO: 21; the polynucleotide SEQ ID NO: 162 encoding the light chain sequence of SEQ ID
NO: 22; the polynucleotide SEQ ID NO: 163 encoding the heavy chain variable sequence
of SEQ ID NO: 23; the polynucleotide SEQ ID NO: 164 encoding the heavy chain
sequence of SEQ ID NO: 24; polynucleotides encoding the complementarity-determining
regions (SEQ ID NO: 165; SEQ ID NO: 166; and SEQ ID NO: 167) of the light chain
variable sequence of SEQ ID NO: 21 or the light chain sequence of SEQ ID NO: 22; and
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 168;
SEQ ID NO: 169; and SEQ ID NO: 170) of the heavy chain variable sequence of SEQ ID
NO: 23 or the heavy chain sequence of SEQ ID NO: 24.
In a preferred embodiment described herein, polynucleotides comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for CGRP. With respect to antibody Ab3, the
polynucleotides encoding the full length Ab3 antibody comprise, or alternatively consist of,
the polynucleotide SEQ ID NO: 162 encoding the light chain sequence of SEQ ID NO: 22
and the polynucleotide SEQ ID NO: 164 encoding the heavy chain sequence of SEQ ID
NO: 24.
Another embodiment described herein contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO,
NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as the
yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab3 following expression of the full-length
polynucleotides in a suitable host. In another embodiment described herein, anti-CGRP
antibodies such as Ab3 or Fab fragments thereof may be produced via expression of Ab3
polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect,
or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia)
and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia
pastoris.
Antibody Ab4
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment described herein,
polynucleotides comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 31:
CAAGTGCTGACCCAGACTCCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATCATAACACCTACCT
GGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAACAACTGATCTATGATG
CATCCACTCTGGCGTCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGG
ACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTAACGATGCTGCCGCTTAC
TACTGTCTGGGCAGTTATGATTGTACTAATGGTGATTGTTTTGTTTTCGGCGGA
GGGACCGAGGTGGTGGTCAAACGT (SEQ ID NO: 171).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the light chain polypeptide
sequence of SEQ ID NO: 32:
CAAGTGCTGACCCAGACTCCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATCATAACACCTACCT
GGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAACAACTGATCTATGATG
CATCCACTCTGGCGTCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGG
ACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTAACGATGCTGCCGCTTAC
TACTGTCTGGGCAGTTATGATTGTACTAATGGTGATTGTTTTGTTTTCGGCGGA
GGGACCGAGGTGGTGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 172).
In another embodiment described herein, polynucleotides comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 33:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGTTCCGTCTCTGGCATCGACCTCAGTGGCTACTACATGAA
CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTGGTA
TTAATGGTGCCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCC
AAAACCTCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGA
CACGGCCACCTATTTCTGTGCCAGAGGGGACATCTGGGGCCCGGGCACCCTCG
TCACCGTCTCGAGC (SEQ ID NO: 173).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the heavy chain polypeptide
sequence of SEQ ID NO: 34:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGAC
ACTCACCTGTTCCGTCTCTGGCATCGACCTCAGTGGCTACTACATGAACTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTGGTATTAATG
GTGCCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACC
TCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGC
CACCTATTTCTGTGCCAGAGGGGACATCTGGGGCCCGGGCACCCTCGTCACCG
TCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCA
AGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA
CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGA
CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT
CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG
TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG
GGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC
ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC
CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG
AACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC
AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA
CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID
NO: 174).
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 175; SEQ ID NO: 176; and SEQ ID
NO: 177 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID
NO: 31 or the light chain sequence of SEQ ID NO: 32.
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 178; SEQ ID NO: 179; and SEQ ID
NO: 180 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID
NO: 33 or the heavy chain sequence of SEQ ID NO: 34.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment described herein, polynucleotides encoding antibody fragments
having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or
more, including all of the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 171 encoding the light chain variable sequence of SEQ ID
NO: 31; the polynucleotide SEQ ID NO: 172 encoding the light chain sequence of SEQ ID
NO: 32; the polynucleotide SEQ ID NO: 173 encoding the heavy chain variable sequence
of SEQ ID NO: 33; the polynucleotide SEQ ID NO: 174 encoding the heavy chain
sequence of SEQ ID NO: 34; polynucleotides encoding the complementarity-determining
regions (SEQ ID NO: 175; SEQ ID NO: 176; and SEQ ID NO: 177) of the light chain
variable sequence of SEQ ID NO: 31 or the light chain sequence of SEQ ID NO: 32; and
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 178;
SEQ ID NO: 179; and SEQ ID NO: 180) of the heavy chain variable sequence of SEQ ID
NO: 33 or the heavy chain sequence of SEQ ID NO: 34.
In a preferred embodiment described herein, polynucleotides comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for CGRP. With respect to antibody Ab4, the
polynucleotides encoding the full length Ab4 antibody comprise, or alternatively consist of,
the polynucleotide SEQ ID NO: 172 encoding the light chain sequence of SEQ ID NO: 32
and the polynucleotide SEQ ID NO: 174 encoding the heavy chain sequence of SEQ ID
NO: 34.
Another embodiment described herein contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO,
NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as the
yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic
digestion (e.g., papain) of Ab4 following expression of the full-length polynucleotides in a
suitable host. In another embodiment described herein, anti-CGRP antibodies such as Ab4
or Fab fragments thereof may be produced via expression of Ab4 polynucleotides in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab5
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment described herein,
polynucleotides comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 41:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATCATAACACCTACCT
GGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATGATG
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTACTAATGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 181).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the light chain polypeptide
sequence of SEQ ID NO: 42:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATCATAACACCTACCT
GGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATGATG
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTACTAATGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 182).
In another embodiment described herein, polynucleotides comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 43:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGAATCGACCTCAGTGGCTACTACAT
GAACTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTG
GTATTAATGGTGCCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 183).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the heavy chain polypeptide
sequence of SEQ ID NO: 44:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGAATCGACCTCAGTGGCTACTACATGAACTG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTGGTATTA
ATGGTGCCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATC
TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG
GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCG
TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 184).
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 185; SEQ ID NO: 186; and SEQ ID
NO: 187 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID
NO: 41 or the light chain sequence of SEQ ID NO: 42.
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 188; SEQ ID NO: 189; and SEQ ID
NO: 190 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID
NO: 43 or the heavy chain sequence of SEQ ID NO: 44.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment described herein, polynucleotides encoding antibody fragments
having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or
more, including all of the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 181 encoding the light chain variable sequence of SEQ ID
NO: 41; the polynucleotide SEQ ID NO: 182 encoding the light chain sequence of SEQ ID
NO: 42; the polynucleotide SEQ ID NO: 183 encoding the heavy chain variable sequence
of SEQ ID NO: 43; the polynucleotide SEQ ID NO: 184 encoding the heavy chain
sequence of SEQ ID NO: 44; polynucleotides encoding the complementarity-determining
regions (SEQ ID NO: 185; SEQ ID NO: 186; and SEQ ID NO: 187) of the light chain
variable sequence of SEQ ID NO: 41 or the light chain sequence of SEQ ID NO: 42; and
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 188;
SEQ ID NO: 189; and SEQ ID NO: 190) of the heavy chain variable sequence of SEQ ID
NO: 43 or the heavy chain sequence of SEQ ID NO: 44.
In a preferred embodiment described herein, polynucleotides comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for CGRP. With respect to antibody Ab5, the
polynucleotides encoding the full length Ab5 antibody comprise, or alternatively consist of,
the polynucleotide SEQ ID NO: 182 encoding the light chain sequence of SEQ ID NO: 42
and the polynucleotide SEQ ID NO: 184 encoding the heavy chain sequence of SEQ ID
NO: 44.
Another embodiment described herein contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO,
NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as the
yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab5 following expression of the full-length
polynucleotides in a suitable host. In another embodiment described herein, anti-CGRP
antibodies such as Ab5 or Fab fragments thereof may be produced via expression of Ab5
polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect,
or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia)
and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia
pastoris.
Antibody Ab6
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment described herein,
polynucleotides comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 51:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATCATAACACCTACCT
GGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATGATG
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTACTAATGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 191).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the light chain polypeptide
sequence of SEQ ID NO: 52:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATCATAACACCTACCT
GGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATGATG
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTACTAATGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 192).
In another embodiment described herein, polynucleotides comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 53:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGAATCGACCTCAGTGGCTACTACAT
GAACTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTG
GTATTAATGGTGCCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 193).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the heavy chain polypeptide
sequence of SEQ ID NO: 54:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGAATCGACCTCAGTGGCTACTACATGAACTG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTGGTATTA
ATGGTGCCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACGCGAGAGTTGAGCCCAAATCT
TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG
ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG
GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG
CCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGT
CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 194).
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 195; SEQ ID NO: 196; and SEQ ID
NO: 197 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID
NO: 51 or the light chain sequence of SEQ ID NO: 52.
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 198; SEQ ID NO: 199; and SEQ ID
NO: 200 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID
NO: 53 or the heavy chain sequence of SEQ ID NO: 54.
The present disclosurealso contemplates polynucleotide sequences including one
or more of the polynucleotide sequences encoding antibody fragments described herein. In
one embodiment described herein, polynucleotides encoding antibody fragments having
binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 191 encoding the light chain variable sequence of SEQ ID
NO: 51; the polynucleotide SEQ ID NO: 192 encoding the light chain sequence of SEQ ID
NO: 52; the polynucleotide SEQ ID NO: 193 encoding the heavy chain variable sequence
of SEQ ID NO: 53; the polynucleotide SEQ ID NO: 194 encoding the heavy chain
sequence of SEQ ID NO: 54; polynucleotides encoding the complementarity-determining
regions (SEQ ID NO: 195; SEQ ID NO: 196; and SEQ ID NO: 197) of the light chain
variable sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 52; and
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 198;
SEQ ID NO: 199; and SEQ ID NO: 200) of the heavy chain variable sequence of SEQ ID
NO: 53 or the heavy chain sequence of SEQ ID NO: 54.
In a preferred embodiment described herein, polynucleotides comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for CGRP. With respect to antibody Ab6, the
polynucleotides encoding the full length Ab6 antibody comprise, or alternatively consist of,
the polynucleotide SEQ ID NO: 192 encoding the light chain sequence of SEQ ID NO: 52
and the polynucleotide SEQ ID NO: 194 encoding the heavy chain sequence of SEQ ID
NO: 54.
Another embodiment described herein contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO,
NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as the
yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein described herein (infra), Fab fragments may be produced by
enzymatic digestion (e.g., papain) of Ab6 following expression of the full-length
polynucleotides in a suitable host. In another embodiment described herein, anti-CGRP
antibodies such as Ab6 or Fab fragments thereof may be produced via expression of Ab6
polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect,
or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia)
and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia
pastoris.
Antibody Ab7
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment described herein,
polynucleotides comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 61:
CAAGTGCTGACCCAGACTGCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAATTACAACTACCT
TGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCTCATCGCGATTCAAAGGCAGTGGATCTGGG
ACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTAC
TACTGTCTAGGCAGTTATGACTGTAGTACTGGTGATTGTTTTGTTTTCGGCGGA
GGGACCGAGGTGGTGGTCAAACGT (SEQ ID NO: 201).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the light chain polypeptide
sequence of SEQ ID NO: 62:
CAAGTGCTGACCCAGACTGCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAATTACAACTACCT
TGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCTCATCGCGATTCAAAGGCAGTGGATCTGGG
ACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTAC
TACTGTCTAGGCAGTTATGACTGTAGTACTGGTGATTGTTTTGTTTTCGGCGGA
GGGACCGAGGTGGTGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 202).
In another embodiment described herein, polynucleotides comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 63:
CAGGAGCAGCTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGA
CATCCCTGACACTCACCTGCACCGTCTCTGGAATCGACCTCAGTAACCACTACA
TGCAATGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCGTT
GGTATTAATGGTCGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCAT
CTCCAGAACCTCGTCGACCACGGTGGATCTGAAAATGACCAGGCTGACAACCG
AGGACACGGCCACCTATTTCTGTGCCAGAGGGGACATCTGGGGCCCAGGCACC
CTGGTCACCGTCTCGAGC (SEQ ID NO: 203).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the heavy chain polypeptide
sequence of SEQ ID NO: 64:
CAGGAGCAGCTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACATCCCT
GACACTCACCTGCACCGTCTCTGGAATCGACCTCAGTAACCACTACATGCAAT
GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCGTTGGTATT
AATGGTCGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAG
AACCTCGTCGACCACGGTGGATCTGAAAATGACCAGGCTGACAACCGAGGACA
CGGCCACCTATTTCTGTGCCAGAGGGGACATCTGGGGCCCAGGCACCCTGGTC
ACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC
TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCG
TGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCG
TGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG
AATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGA
CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGT
CAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC
CGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA
AGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC
GAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAA
CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGT
GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC
GTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAG
AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT
GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ
ID NO: 204).
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 205; SEQ ID NO: 206; and SEQ ID
NO: 207 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID
NO: 61 or the light chain sequence of SEQ ID NO: 62.
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 208; SEQ ID NO: 209; and SEQ ID
NO: 210 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID
NO: 63 or the heavy chain sequence of SEQ ID NO: 64.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment described herein, polynucleotides encoding antibody fragments
having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or
more, including all of the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 201 encoding the light chain variable sequence of SEQ ID
NO: 61; the polynucleotide SEQ ID NO: 202 encoding the light chain sequence of SEQ ID
NO: 62; the polynucleotide SEQ ID NO: 203 encoding the heavy chain variable sequence
of SEQ ID NO: 63; the polynucleotide SEQ ID NO: 204 encoding the heavy chain
sequence of SEQ ID NO: 64; polynucleotides encoding the complementarity-determining
regions (SEQ ID NO: 205; SEQ ID NO: 206; and SEQ ID NO: 207) of the light chain
variable sequence of SEQ ID NO: 61 or the light chain sequence of SEQ ID NO: 62; and
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 208;
SEQ ID NO: 209; and SEQ ID NO: 210) of the heavy chain variable sequence of SEQ ID
NO: 63 or the heavy chain sequence of SEQ ID NO: 64.
In a preferred embodiment described herein, polynucleotides comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for CGRP. With respect to antibody Ab7, the
polynucleotides encoding the full length Ab7 antibody comprise, or alternatively consist of,
the polynucleotide SEQ ID NO: 202 encoding the light chain sequence of SEQ ID NO: 62
and the polynucleotide SEQ ID NO: 204 encoding the heavy chain sequence of SEQ ID
NO: 64.
Another embodiment described herein contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO,
NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as the
yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic
digestion (e.g., papain) of Ab7 following expression of the full-length polynucleotides in a
suitable host. In another embodiment described herein, anti-CGRP antibodies such as Ab7
or Fab fragments thereof may be produced via expression of Ab7 polynucleotides in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab8
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment described herein,
polynucleotides comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 71:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTACAATTACAACTACCTT
GCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTAC
ATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGAC
AGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTA
CTGTCTGGGCAGTTATGATTGTAGTACTGGTGATTGTTTTGTTTTCGGCGGAGG
AACCAAGGTGGAAATCAAACGT (SEQ ID NO: 211).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the light chain polypeptide
sequence of SEQ ID NO: 72:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTACAATTACAACTACCTT
GCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTAC
ATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGAC
AGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTA
CTGTCTGGGCAGTTATGATTGTAGTACTGGTGATTGTTTTGTTTTCGGCGGAGG
AACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCC
GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAA
TAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC
AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCAC
CTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC
AAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAA
GAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 212).
In another embodiment described herein, polynucleotides comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 73:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGAATCGACCTCAGTAACCACTACAT
GCAATGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCGTTG
GTATCAATGGTCGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 213).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the heavy chain polypeptide
sequence of SEQ ID NO: 74:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGAATCGACCTCAGTAACCACTACATGCAATG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCGTTGGTATCA
ATGGTCGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATC
TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG
GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCG
TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 214).
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 215; SEQ ID NO: 216; and SEQ ID
NO: 217 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID
NO: 71 or the light chain sequence of SEQ ID NO: 72.
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 218; SEQ ID NO: 219; and SEQ ID
NO: 220 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID
NO: 73 or the heavy chain sequence of SEQ ID NO: 74.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment described herein, polynucleotides encoding antibody fragments
having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or
more, including all of the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 211 encoding the light chain variable sequence of SEQ ID
NO: 71; the polynucleotide SEQ ID NO: 212 encoding the light chain sequence of SEQ ID
NO: 72; the polynucleotide SEQ ID NO: 213 encoding the heavy chain variable sequence
of SEQ ID NO: 73; the polynucleotide SEQ ID NO: 214 encoding the heavy chain
sequence of SEQ ID NO: 74; polynucleotides encoding the complementarity-determining
regions (SEQ ID NO: 215; SEQ ID NO: 216; and SEQ ID NO: 217) of the light chain
variable sequence of SEQ ID NO: 71 or the light chain sequence of SEQ ID NO: 72; and
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 218;
SEQ ID NO: 219; and SEQ ID NO: 220) of the heavy chain variable sequence of SEQ ID
NO: 73 or the heavy chain sequence of SEQ ID NO: 74.
In a preferred embodiment described herein, polynucleotides comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for CGRP. With respect to antibody Ab8, the
polynucleotides encoding the full length Ab8 antibody comprise, or alternatively consist of,
the polynucleotide SEQ ID NO: 212 encoding the light chain sequence of SEQ ID NO: 72
and the polynucleotide SEQ ID NO: 214 encoding the heavy chain sequence of SEQ ID
NO: 74.
Another embodiment described herein contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO,
NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as the
yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic
digestion (e.g., papain) of Ab8 following expression of the full-length polynucleotides in a
suitable host. In another embodiment described herein, anti-CGRP antibodies such as Ab8
or Fab fragments thereof may be produced via expression of Ab8 polynucleotides in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab9
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment described herein,
polynucleotides comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 81:
CAAGTGCTGACCCAGACTCCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTTATAATAACAACTACC
TAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCT
ACGTCCACTCTGGCATCTGGGGTCTCATCGCGATTCAGAGGCAGTGGATCTGG
GACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTT
ACTACTGTCTAGGCAGTTATGATTGTAGTCGTGGTGATTGTTTTGTTTTCGGCG
GAGGGACCGAGGTGGTGGTCAAACGT (SEQ ID NO: 221).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the light chain polypeptide
sequence of SEQ ID NO: 82:
CAAGTGCTGACCCAGACTCCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTTATAATAACAACTACC
TAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCT
ACGTCCACTCTGGCATCTGGGGTCTCATCGCGATTCAGAGGCAGTGGATCTGG
GACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTT
ACTACTGTCTAGGCAGTTATGATTGTAGTCGTGGTGATTGTTTTGTTTTCGGCG
GAGGGACCGAGGTGGTGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATC
TTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTG
CTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGC
CCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGAC
AGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGA
AACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTC
ACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 222).
In another embodiment described herein, polynucleotides comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 83:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGTCTCTGGAATCGGCCTCAGTAGCTACTACATGCA
GTGGGTCCGCCAGTCTCCAGGGAGGGGGCTGGAATGGATCGGAGTCATTGGTA
GTGATGGTAAGACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCC
AAGACCTCGTCGACCACGGTGGATCTGAGAATGGCCAGTCTGACAACCGAGGA
CACGGCCACCTATTTCTGTACCAGAGGGGACATCTGGGGCCCGGGGACCCTCG
TCACCGTCTCGAGC (SEQ ID NO: 223).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the heavy chain polypeptide
sequence of SEQ ID NO: 84:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGAC
ACTCACCTGCACAGTCTCTGGAATCGGCCTCAGTAGCTACTACATGCAGTGGGT
CCGCCAGTCTCCAGGGAGGGGGCTGGAATGGATCGGAGTCATTGGTAGTGATG
GTAAGACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAGACC
TCGTCGACCACGGTGGATCTGAGAATGGCCAGTCTGACAACCGAGGACACGGC
CACCTATTTCTGTACCAGAGGGGACATCTGGGGCCCGGGGACCCTCGTCACCG
TCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCA
AGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA
CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGA
CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT
CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG
TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG
GGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC
ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC
CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG
AACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC
AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA
CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID
NO: 224).
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 225; SEQ ID NO: 226; and SEQ ID
NO: 227 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID
NO: 81 or the light chain sequence of SEQ ID NO: 82.
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 228; SEQ ID NO: 229; and SEQ ID
NO: 230 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID
NO: 83 or the heavy chain sequence of SEQ ID NO: 84.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment described herein, polynucleotides encoding antibody fragments
having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or
more, including all of the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 221 encoding the light chain variable sequence of SEQ ID
NO: 81; the polynucleotide SEQ ID NO: 222 encoding the light chain sequence of SEQ ID
NO: 82; the polynucleotide SEQ ID NO: 223 encoding the heavy chain variable sequence
of SEQ ID NO: 83; the polynucleotide SEQ ID NO: 224 encoding the heavy chain
sequence of SEQ ID NO: 84; polynucleotides encoding the complementarity-determining
regions (SEQ ID NO: 225; SEQ ID NO: 226; and SEQ ID NO: 227) of the light chain
variable sequence of SEQ ID NO: 81 or the light chain sequence of SEQ ID NO: 82; and
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 228;
SEQ ID NO: 229; and SEQ ID NO: 230) of the heavy chain variable sequence of SEQ ID
NO: 83 or the heavy chain sequence of SEQ ID NO: 84.
In a preferred embodiment described herein, polynucleotides comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for CGRP. With respect to antibody Ab9, the
polynucleotides encoding the full length Ab9 antibody comprise, or alternatively consist of,
the polynucleotide SEQ ID NO: 222 encoding the light chain sequence of SEQ ID NO: 82
and the polynucleotide SEQ ID NO: 224 encoding the heavy chain sequence of SEQ ID
NO: 84.
Another embodiment described herein contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO,
NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as the
yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic
digestion (e.g., papain) of Ab9 following expression of the full-length polynucleotides in a
suitable host. In another embodiment described herein, anti-CGRP antibodies such as Ab9
or Fab fragments thereof may be produced via expression of Ab9 polynucleotides in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab10
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment described herein,
polynucleotides comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 91:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTTACAATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTAGTCGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 231).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the light chain polypeptide
sequence of SEQ ID NO: 92:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTTACAATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTAGTCGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 232).
In another embodiment described herein, polynucleotides comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 93:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGAATCGGCCTCAGTAGCTACTACAT
GCAATGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTG
GTAGTGATGGTAAGACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTACCAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 233).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the heavy chain polypeptide
sequence of SEQ ID NO: 94:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGAATCGGCCTCAGTAGCTACTACATGCAATG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTGGTAGTG
ATGGTAAGACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTACCAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATC
TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG
GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCG
TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 234).
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 235; SEQ ID NO: 236; and SEQ ID
NO: 237 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID
NO: 91 or the light chain sequence of SEQ ID NO: 92.
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 238; SEQ ID NO: 239; and SEQ ID
NO:240 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID
NO: 93 or the heavy chain sequence of SEQ ID NO: 94.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment described herein, polynucleotides encoding antibody fragments
having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or
more, including all of the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 231 encoding the light chain variable sequence of SEQ ID
NO: 91; the polynucleotide SEQ ID NO: 232 encoding the light chain sequence of SEQ ID
NO: 92; the polynucleotide SEQ ID NO: 233 encoding the heavy chain variable sequence
of SEQ ID NO: 93; the polynucleotide SEQ ID NO: 234 encoding the heavy chain
sequence of SEQ ID NO: 94; polynucleotides encoding the complementarity-determining
regions (SEQ ID NO: 235; SEQ ID NO: 236; and SEQ ID NO: 237) of the light chain
variable sequence of SEQ ID NO: 91 or the light chain sequence of SEQ ID NO: 92; and
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 238;
SEQ ID NO: 239; and SEQ ID NO: 240) of the heavy chain variable sequence of SEQ ID
NO: 93 or the heavy chain sequence of SEQ ID NO: 94.
In a preferred embodiment described herein, polynucleotides comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for CGRP. With respect to antibody Ab10, the
polynucleotides encoding the full length Ab10 antibody comprise, or alternatively consist
of, the polynucleotide SEQ ID NO: 232 encoding the light chain sequence of SEQ ID NO:
92 and the polynucleotide SEQ ID NO: 234 encoding the heavy chain sequence of SEQ ID
NO: 94.
Another embodiment described herein contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO,
NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as the
yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic
digestion (e.g., papain) of Ab10 following expression of the full-length polynucleotides in
a suitable host. In another embodiment described herein, anti-CGRP antibodies such as
Ab10 or Fab fragments thereof may be produced via expression of Ab10 polynucleotides in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab11
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment described herein,
polynucleotides comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 101:
CAGGTGCTGACCCAGACTGCATCCCCCGTGTCTCCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCGGGCCAGTCAGAGTGTTTATTATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGG
ACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTAC
TACTGTCTAGGCAGTTATGATTGTAGTAATGGTGATTGTTTTGTTTTCGGCGGA
GGGACCGAGGTGGTGGTCAAACGT (SEQ ID NO: 241).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the light chain polypeptide
sequence of SEQ ID NO: 102:
CAGGTGCTGACCCAGACTGCATCCCCCGTGTCTCCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCGGGCCAGTCAGAGTGTTTATTATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGG
ACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTAC
TACTGTCTAGGCAGTTATGATTGTAGTAATGGTGATTGTTTTGTTTTCGGCGGA
GGGACCGAGGTGGTGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 242).
In another embodiment described herein, polynucleotides comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 103:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGAGGATC
CCTGACACTCACCTGCACAGTCTCTGGAATCGACGTCACTAACTACTATATGCA
ATGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTGGTG
TGAATGGTAAGAGATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCC
AAAACCTCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGA
CACGGCCACCTATTTCTGTGCCAGAGGCGACATCTGGGGCCCGGGGACCCTCG
TCACCGTCTCGAGC (SEQ ID NO: 243).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the heavy chain polypeptide
sequence of SEQ ID NO: 104:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGAGGATCCCTGAC
ACTCACCTGCACAGTCTCTGGAATCGACGTCACTAACTACTATATGCAATGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTGGTGTGAATG
GTAAGAGATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACC
TCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGC
CACCTATTTCTGTGCCAGAGGCGACATCTGGGGCCCGGGGACCCTCGTCACCG
TCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCA
AGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA
CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGA
CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT
CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG
TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG
GGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC
ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC
CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG
AACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC
AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA
CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID
NO: 244).
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 245; SEQ ID NO: 246; and SEQ ID
NO: 247 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID
NO: 101 or the light chain sequence of SEQ ID NO: 102.
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 248; SEQ ID NO: 249; and SEQ ID
NO: 250 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID
NO: 103 or the heavy chain sequence of SEQ ID NO: 104.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment described herein, polynucleotides encoding antibody fragments
having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or
more, including all of the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 241 encoding the light chain variable sequence of SEQ ID
NO: 101; the polynucleotide SEQ ID NO: 242 encoding the light chain sequence of SEQ
ID NO: 102; the polynucleotide SEQ ID NO: 243 encoding the heavy chain variable
sequence of SEQ ID NO: 103; the polynucleotide SEQ ID NO: 244 encoding the heavy
chain sequence of SEQ ID NO: 104; polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 245; SEQ ID NO: 246; and SEQ ID NO: 247) of the
light chain variable sequence of SEQ ID NO: 101 or the light chain sequence of SEQ ID
NO: 102; and polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 248; SEQ ID NO: 249; and SEQ ID NO: 250) of the heavy chain variable sequence of
SEQ ID NO: 103 or the heavy chain sequence of SEQ ID NO: 104.
In a preferred embodiment described herein, polynucleotides comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for CGRP. With respect to antibody Ab11, the
polynucleotides encoding the full length Ab11 antibody comprise, or alternatively consist
of, the polynucleotide SEQ ID NO: 242 encoding the light chain sequence of SEQ ID NO:
102 and the polynucleotide SEQ ID NO: 244 encoding the heavy chain sequence of SEQ
ID NO: 104.
Another embodiment described herein contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO,
NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as the
yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic
digestion (e.g., papain) of Ab11 following expression of the full-length polynucleotides in
a suitable host. In another embodiment described herein, anti-CGRP antibodies such as
Ab11 or Fab fragments thereof may be produced via expression of Ab11 polynucleotides in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab12
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment described herein,
polynucleotides comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 111:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCGGGCCAGTCAGAGTGTTTACTATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTAGTAATGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 251).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the light chain polypeptide
sequence of SEQ ID NO: 112:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCGGGCCAGTCAGAGTGTTTACTATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTAGTAATGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 252).
In another embodiment described herein, polynucleotides comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 113:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGAATCGACGTCACTAACTACTACAT
GCAATGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTG
GTGTGAATGGTAAGAGATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTGCCAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 253).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the heavy chain polypeptide
sequence of SEQ ID NO: 114:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGAATCGACGTCACTAACTACTACATGCAATG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTGGTGTGA
ATGGTAAGAGATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTGCCAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATC
TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG
GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCG
TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 254).
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 255; SEQ ID NO: 256; and SEQ ID
NO: 257 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID
NO: 111 or the light chain sequence of SEQ ID NO: 112.
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 258; SEQ ID NO: 259; and SEQ ID
NO: 260 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID
NO: 113 or the heavy chain sequence of SEQ ID NO: 114.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment described herein, polynucleotides encoding antibody fragments
having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or
more, including all of the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 251 encoding the light chain variable sequence of SEQ ID
NO: 111; the polynucleotide SEQ ID NO: 252 encoding the light chain sequence of SEQ
ID NO: 112; the polynucleotide SEQ ID NO: 253 encoding the heavy chain variable
sequence of SEQ ID NO: 113; the polynucleotide SEQ ID NO: 254 encoding the heavy
chain sequence of SEQ ID NO: 114; polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 255; SEQ ID NO: 256; and SEQ ID NO: 257) of the
light chain variable sequence of SEQ ID NO: 111 or the light chain sequence of SEQ ID
NO: 112; and polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 258; SEQ ID NO: 259; and SEQ ID NO: 260) of the heavy chain variable sequence of
SEQ ID NO: 113 or the heavy chain sequence of SEQ ID NO: 114.
In a preferred embodiment described herein, polynucleotides comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for CGRP. With respect to antibody Ab12, the
polynucleotides encoding the full length Ab12 antibody comprise, or alternatively consist
of, the polynucleotide SEQ ID NO: 252 encoding the light chain sequence of SEQ ID NO:
112 and the polynucleotide SEQ ID NO: 254 encoding the heavy chain sequence of SEQ
ID NO: 114.
Another embodiment described herein contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO,
NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as the
yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic
digestion (e.g., papain) of Ab12 following expression of the full-length polynucleotides in
a suitable host. In another embodiment described herein, anti-CGRP antibodies such as
Ab12 or Fab fragments thereof may be produced via expression of Ab12 polynucleotides in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab13
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment described herein,
polynucleotides comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 121:
GCCATCGTGATGACCCAGACTCCATCTTCCAAGTCTGTCCCTGTGGGA
GACACAGTCACCATCAATTGCCAGGCCAGTGAGAGTCTTTATAATAACAACGC
CTTGGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTATG
ATGCATCCAAACTGGCATCTGGGGTCCCATCGCGGTTCAGTGGCGGTGGGTCT
GGGACACAGTTCACTCTCACCATCAGTGGCGTGCAGTGTGACGATGCTGCCAC
TTACTACTGTGGAGGCTACAGAAGTGATAGTGTTGATGGTGTTGCTTTCGCCGG
AGGGACCGAGGTGGTGGTCAAACGT (SEQ ID NO: 261).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the light chain polypeptide
sequence of SEQ ID NO: 122:
GCCATCGTGATGACCCAGACTCCATCTTCCAAGTCTGTCCCTGTGGGA
GACACAGTCACCATCAATTGCCAGGCCAGTGAGAGTCTTTATAATAACAACGC
CTTGGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTATG
ATGCATCCAAACTGGCATCTGGGGTCCCATCGCGGTTCAGTGGCGGTGGGTCT
GGGACACAGTTCACTCTCACCATCAGTGGCGTGCAGTGTGACGATGCTGCCAC
TTACTACTGTGGAGGCTACAGAAGTGATAGTGTTGATGGTGTTGCTTTCGCCGG
AGGGACCGAGGTGGTGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCT
TCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGC
TGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC
CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACA
GCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAA
ACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCA
CAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 262).
In another embodiment described herein, polynucleotides comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 123:
CAGTCGGTGGAGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGAT
CCCTGACACTCACCTGCACAGCCTCTGGATTCGACTTCAGTAGCAATGCAATGT
GGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATGCATTTAC
AATGGTGATGGCAGCACATACTACGCGAGCTGGGTGAATGGCCGATTCTCCAT
CTCCAAAACCTCGTCGACCACGGTGACTCTGCAACTGAATAGTCTGACAGTCG
CGGACACGGCCACGTATTATTGTGCGAGAGATCTTGACTTGTGGGGCCCGGGC
ACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 263).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the heavy chain polypeptide
sequence of SEQ ID NO: 124:
CAGTCGGTGGAGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGATCCCTGAC
ACTCACCTGCACAGCCTCTGGATTCGACTTCAGTAGCAATGCAATGTGGTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATGCATTTACAATGGTG
ATGGCAGCACATACTACGCGAGCTGGGTGAATGGCCGATTCTCCATCTCCAAA
ACCTCGTCGACCACGGTGACTCTGCAACTGAATAGTCTGACAGTCGCGGACAC
GGCCACGTATTATTGTGCGAGAGATCTTGACTTGTGGGGCCCGGGCACCCTCGT
CACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC
CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACT
ACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC
GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC
GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGT
GAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCT
TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG
ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG
GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG
CCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGT
CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 264).
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 265; SEQ ID NO: 266; and SEQ ID
NO: 267 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID
NO: 121 or the light chain sequence of SEQ ID NO: 122.
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 268; SEQ ID NO: 269; and SEQ ID
NO: 270 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID
NO: 123 or the heavy chain sequence of SEQ ID NO: 124.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment described herein, polynucleotides encoding antibody fragments
having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or
more, including all of the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 261 encoding the light chain variable sequence of SEQ ID
NO: 121; the polynucleotide SEQ ID NO: 262 encoding the light chain sequence of SEQ
ID NO: 122; the polynucleotide SEQ ID NO: 263 encoding the heavy chain variable
sequence of SEQ ID NO: 123; the polynucleotide SEQ ID NO: 264 encoding the heavy
chain sequence of SEQ ID NO: 124; polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 265; SEQ ID NO: 266; and SEQ ID NO: 267) of the
light chain variable sequence of SEQ ID NO: 121 or the light chain sequence of SEQ ID
NO: 122; and polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 268; SEQ ID NO: 269; and SEQ ID NO: 270) of the heavy chain variable sequence of
SEQ ID NO: 123 or the heavy chain sequence of SEQ ID NO: 124.
In a preferred embodiment described herein, polynucleotides comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for CGRP. With respect to antibody Ab13, the
polynucleotides encoding the full length Ab13 antibody comprise, or alternatively consist
of, the polynucleotide SEQ ID NO: 262 encoding the light chain sequence of SEQ ID NO:
122 and the polynucleotide SEQ ID NO: 264 encoding the heavy chain sequence of SEQ
ID NO: 124.
Another embodiment described herein contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO,
NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as the
yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic
digestion (e.g., papain) of Ab13 following expression of the full-length polynucleotides in
a suitable host. In another embodiment described herein, anti-CGRP antibodies such as
Ab13 or Fab fragments thereof may be produced via expression of Ab13 polynucleotides in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab14
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment described herein,
polynucleotides comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 131:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTTACAATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTAGTCGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 271).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the light chain polypeptide
sequence of SEQ ID NO: 132:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTTACAATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTAGTCGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 272).
In another embodiment described herein, polynucleotides comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 133:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGAATCGGCCTCAGTAGCTACTACAT
GCAATGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTG
GTAGTGATGGTAAGACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTACCAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 273).
In one embodiment described herein, polynucleotides comprise, or alternatively
consist of, the following polynucleotide sequence encoding the heavy chain polypeptide
sequence of SEQ ID NO: 134:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGAATCGGCCTCAGTAGCTACTACATGCAATG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTGGTAGTG
ATGGTAAGACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTACCAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACGCGAGAGTTGAGCCCAAATCT
TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG
ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG
GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG
CCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGT
CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 274).
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 275; SEQ ID NO: 276; and SEQ ID
NO: 277 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID
NO: 131 or the light chain sequence of SEQ ID NO: 132.
In a further embodiment described herein, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 278; SEQ ID NO: 279; and SEQ ID
NO: 280 which correspond to polynucleotides encoding the complementarity-determining
regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID
NO: 133 or the heavy chain sequence of SEQ ID NO: 134.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment described herein, polynucleotides encoding antibody fragments
having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or
more, including all of the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 271 encoding the light chain variable sequence of SEQ ID
NO: 131; the polynucleotide SEQ ID NO: 272 encoding the light chain sequence of SEQ
ID NO: 132; the polynucleotide SEQ ID NO: 273 encoding the heavy chain variable
sequence of SEQ ID NO: 133; the polynucleotide SEQ ID NO: 274 encoding the heavy
chain sequence of SEQ ID NO: 134; polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 275; SEQ ID NO: 276; and SEQ ID NO: 277) of the
light chain variable sequence of SEQ ID NO: 131 or the light chain sequence of SEQ ID
NO: 132; and polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 278; SEQ ID NO: 279; and SEQ ID NO: 280) of the heavy chain variable sequence of
SEQ ID NO: 133 or the heavy chain sequence of SEQ ID NO: 134.
In a preferred embodiment described herein, polynucleotides comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for CGRP. With respect to antibody Ab14, the
polynucleotides encoding the full length Ab14 antibody comprise, or alternatively consist
of, the polynucleotide SEQ ID NO: 272 encoding the light chain sequence of SEQ ID NO:
132 and the polynucleotide SEQ ID NO: 274 encoding the heavy chain sequence of SEQ
ID NO: 134.
Another embodiment described herein contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO,
NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as the
yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic
digestion (e.g., papain) of Ab14 following expression of the full-length polynucleotides in
a suitable host. In another embodiment described herein, anti-CGRP antibodies such as
Ab14 or Fab fragments thereof may be produced via expression of Ab14 polynucleotides in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other
yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
In one embodiment, the present disclosure is directed to an isolated
polynucleotide comprising a polynucleotide encoding an anti-CGRP V antibody amino
acid sequence selected from SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113,
123, or 133, or encoding a variant thereof wherein at least one framework residue (FR
residue) has been substituted with an amino acid present at the corresponding position in a
rabbit anti-CGRP antibody V polypeptide or a conservative amino acid substitution.
In another embodiment, the present disclosure is directed to an isolated
polynucleotide comprising the polynucleotide sequence encoding an anti-CGRP V
antibody amino acid sequence of 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, or
131, or encoding a variant thereof wherein at least one framework residue (FR residue) has
been substituted with an amino acid present at the corresponding position in a rabbit anti-
CGRP antibody V polypeptide or a conservative amino acid substitution.
In yet another embodiment, the present disclosure is directed to one or more
heterologous polynucleotides comprising a sequence encoding the polypeptides contained
in SEQ ID NO:1 and SEQ ID NO:3; SEQ ID NO:11 and SEQ ID NO:13; SEQ ID NO:21
and SEQ ID NO:23; SEQ ID NO:31 and SEQ ID NO:33; SEQ ID NO:41 and SEQ ID
NO:43; SEQ ID NO:51 and SEQ ID NO:53, SEQ ID NO:61 and SEQ ID NO:63; SEQ ID
NO:71 and SEQ ID NO:73; SEQ ID NO:81 and SEQ ID NO:83; SEQ ID NO:91 and SEQ
ID NO:93; SEQ ID NO:101 and SEQ ID NO:103; SEQ ID NO:111 and SEQ ID NO:113;
SEQ ID NO:121 and SEQ ID NO:123; or SEQ ID NO:131 and SEQ ID NO:133.
In another embodiment, the present disclosure is directed to an isolated
polynucleotide that expresses a polypeptide containing at least one CDR polypeptide
derived from an anti-CGRP antibody wherein said expressed polypeptide alone specifically
binds CGRP or specifically binds CGRP when expressed in association with another
polynucleotide sequence that expresses a polypeptide containing at least one CDR
polypeptide derived from an anti-CGRP antibody wherein said at least one CDR is selected
from those contained in the V or V polypeptides of SEQ ID NO: 1, 3, 11, 13, 21, 23, 31,
33, 41, 43, 51, 53, 61, 63, 71, 73, 81, 83, 91, 93, 101, 103, 111, 113, 121, 123, 131, or SEQ
ID NO:133.
Host cells and vectors comprising said polynucleotides are also contemplated.
The present disclosure further contemplates vectors comprising the
polynucleotide sequences encoding the variable heavy and light chain polypeptide
sequences, as well as the individual complementarity-determining regions (CDRs, or
hypervariable regions), as set forth herein, as well as host cells comprising said vector
sequences. In one embodiment described herein, the host cell is a yeast cell. In another
embodiment, the yeast host cell belongs to the genus Pichia.
B-cell Screening and Isolation
In one embodiment, the present dislcosure contemplates the preparation and
isolation of a clonal population of antigen-specific B cells that may be used for isolating at
least one CGRP antigen-specific cell, which can be used to produce a monoclonal antibody
against CGRP, which is specific to a desired CGRP antigen, or a nucleic acid sequence
corresponding to such an antibody. Methods of preparing and isolating said clonal
population of antigen-specific B cells are taught, for example, in U.S. patent publication
no. US 2007/0269868 to Carvalho-Jensen et al., the disclosure of which is herein
incorporated by reference in its entirety. Methods of preparing and isolating said clonal
population of antigen-specific B cells are also taught herein in the examples. Methods of
“enriching” a cell population by size or density are known in the art. See, e.g., U.S. Patent
,627,052. These steps can be used in addition to enriching the cell population by antigen-
specificity.
Methods of Humanizing Antibodies
In another embodiment, the present disclosure contemplates methods for
humanizing antibody heavy and light chains. Methods for humanizing antibody heavy and
light chains which may be applied to anti-CGRP antibodies are taught, for example, in U.S.
patent application publication no. US 2009/0022659 to Olson et al., and in U.S. patent no.
7,935,340 to Garcia-Martinez et al., the disclosures of each of which are herein
incorporated by reference in their entireties.
Methods of Producing Antibodies and Fragments thereof
In another embodiment, the present disclosure contemplates methods for
producing anti-CGRP antibodies and fragments thereof. Methods for producing anti-
CGRP antibodies and fragments thereof secreted from polyploidal, preferably diploid or
tetraploid strains of mating competent yeast are taught, for example, in U.S. patent
application publication no. US 2009/0022659 to Olson et al., and in U.S. patent no.
7,935,340 to Garcia-Martinez et al., the disclosures of each of which are herein
incorporated by reference in their entireties.
Other methods of producing antibodies are well known to those of ordinary skill
in the art. For example, methods of producing chimeric antibodies are now well known in
the art (See, for example, U.S. Patent No. 4,816,567 to Cabilly et al.; Morrison et al.,
P.N.A.S. USA, 81:8651-55 (1984); Neuberger, M.S. et al., Nature, 314:268-270 (1985);
Boulianne, G.L. et al., Nature, 312:643-46 (1984), the disclosures of each of which are
herein incorporated by reference in their entireties).
Likewise, other methods of producing humanized antibodies are now well
known in the art (See, for example, U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and
6,180,370 to Queen et al; U.S. Patent Nos. 5,225,539 and 6,548,640 to Winter; U.S. Patent
Nos. 6,054,297, 6,407,213 and 6,639,055 to Carter et al; U.S. Patent No. 6,632,927 to
Adair; Jones, P.T. et al, Nature, 321:522-525 (1986); Reichmann, L., et al, Nature,
332:323-327 (1988); Verhoeyen, M, et al, Science, 239:1534-36 (1988), the disclosures of
each of which are herein incorporated by reference in their entireties).
Antibody polypeptides described herein having CGRP binding specificity may
also be produced by constructing, using conventional techniques well known to those of
ordinary skill in the art, an expression vector containing an operon and a DNA sequence
encoding an antibody heavy chain in which the DNA sequence encoding the CDRs
required for antibody specificity is derived from a non-human cell source, preferably a
rabbit B-cell source, while the DNA sequence encoding the remaining parts of the antibody
chain is derived from a human cell source.
A second expression vector is produced using the same conventional means well
known to those of ordinary skill in the art, said expression vector containing an operon and
a DNA sequence encoding an antibody light chain in which the DNA sequence encoding
the CDRs required for antibody specificity is derived from a non-human cell source,
preferably a rabbit B-cell source, while the DNA sequence encoding the remaining parts of
the antibody chain is derived from a human cell source.
The expression vectors are transfected into a host cell by convention techniques
well known to those of ordinary skill in the art to produce a transfected host cell, said
transfected host cell cultured by conventional techniques well known to those of ordinary
skill in the art to produce said antibody polypeptides.
The host cell may be co-transfected with the two expression vectors described
above, the first expression vector containing DNA encoding an operon and a light chain-
derived polypeptide and the second vector containing DNA encoding an operon and a
heavy chain-derived polypeptide. The two vectors contain different selectable markers, but
preferably achieve substantially equal expression of the heavy and light chain polypeptides.
Alternatively, a single vector may be used, the vector including DNA encoding both the
heavy and light chain polypeptides. The coding sequences for the heavy and light chains
may comprise cDNA, genomic DNA, or both.
The host cells used to express the antibody polypeptides may be either a
bacterial cell such as E. coli, or a eukaryotic cell such as P. pastoris. In one embodiment of
the present disclosure, a mammalian cell of a well-defined type for this purpose, such as a
myeloma cell, a Chinese hamster ovary (CHO) cell line, a NSO cell line, or a HEK293 cell
line may be used.
The general methods by which the vectors may be constructed, transfection
methods required to produce the host cell and culturing methods required to produce the
antibody polypeptides from said host cells all include conventional techniques. Although
preferably the cell line used to produce the antibody is a mammalian cell line, any other
suitable cell line, such as a bacterial cell line such as an E. coli-derived bacterial strain, or a
yeast cell line, may alternatively be used.
Similarly, once produced the antibody polypeptides may be purified according to
standard procedures in the art, such as for example cross-flow filtration, ammonium
sulphate precipitation, affinity column chromatography and the like.
The antibody polypeptides described herein may also be used for the design and
synthesis of either peptide or non-peptide mimetics that would be useful for the same
therapeutic applications as the antibody polypeptides described herein. See, for example,
Saragobi et al, Science, 253:792-795 (1991), the contents of which is herein incorporated
by reference in its entirety.
Screening Assays
The present disclosure also includes screening assays designed to assist in the
identification of diseases and disorders associated with CGRP in patients exhibiting
symptoms of a CGRP associated disease or disorder.
In one embodiment described herein, the anti-CGRP antibodies, or CGRP
binding fragments thereof, are used to detect the presence of CGRP in a biological sample
obtained from a patient exhibiting symptoms of a disease or disorder associated with
CGRP. The presence of CGRP, or elevated levels thereof when compared to pre-disease
levels of CGRP in a comparable biological sample, may be beneficial in diagnosing a
disease or disorder associated with CGRP.
Another embodiment described herein provides a diagnostic or screening assay
to assist in diagnosis of diseases or disorders associated with CGRP in patients exhibiting
symptoms of a CGRP associated disease or disorder identified herein, comprising assaying
the level of CGRP expression in a biological sample from said patient using a post-
translationally modified anti-CGRP antibody or binding fragment thereof. The anti-CGRP
antibody or binding fragment thereof may be post-translationally modified to include a
detectable moiety such as set forth previously in the disclosure.
The CGRP level in the biological sample is determined using a modified anti-
CGRP antibody or binding fragment thereof as set forth herein, and comparing the level of
CGRP in the biological sample against a standard level of CGRP (e.g., the level in normal
biological samples). The skilled clinician would understand that some variability may exist
between normal biological samples, and would take that into consideration when
evaluating results. In one embodiment described herein, the anti-CGRP antibodies may be
used to correlate CGRP expression levels with a particular stage of cancerous development.
One skilled in the art would be able to measure CGRP in numerous subjects in order to
establish ranges of CGRP expression that correspond to clinically defined stages of
cancerous development. These ranges will allow the skilled practitioner to measure CGRP
in a subject diagnosed with a cancer and correlate the levels in each subject with a range
that corresponds to a stage of said cancer. One skilled in the art would understand that by
measuring CGRP in the patient at different intervals, the progression of the cancer can be
determined.
The above-recited assay may also be useful in monitoring a disease or disorder,
where the level of CGRP obtained in a biological sample from a patient believed to have a
CGRP associated disease or disorder is compared with the level of CGRP in prior
biological samples from the same patient, in order to ascertain whether the CGRP level in
said patient has changed with, for example, a treatment regimen.
The present disclosure is also directed to a method of in vivo imaging which
detects the presence of cells which express CGRP comprising administering a
diagnostically effective amount of a diagnostic composition. Said in vivo imaging is useful
for the detection or imaging of CGRP expressing tumors or metastases, for example, and
can be useful as part of a planning regimen for the design of an effective cancer treatment
protocol. The treatment protocol may include, for example, one or more of radiation,
chemotherapy, cytokine therapy, gene therapy, and antibody therapy, as well as an anti-
CGRP antibody or fragment thereof.
Also described is a kit for detecting binding of an anti-CGRP antibody described
herein to CGRP. In particular, the kit may be used to detect the presence of a CGRP
specifically reactive with an anti-CGRP antibody described herein or an immunoreactive
fragment thereof. The kit may also include an antibody bound to a substrate, a secondary
antibody reactive with the antigen and a reagent for detecting a reaction of the secondary
antibody with the antigen. Such a kit may be an ELISA kit and can comprise the substrate,
primary and secondary antibodies when appropriate, and any other necessary reagents such
as detectable moieties, enzyme substrates, and color reagents, for example as described
herein. The diagnostic kit may also be in the form of an immunoblot kit. The diagnostic
kit may also be in the form of a chemiluminescent kit (Meso Scale Discovery,
Gaithersburg, MD). The diagnostic kit may also be a lanthanide-based detection kit
(PerkinElmer, San Jose, CA).
A skilled clinician would understand that a biological sample includes, but is not
limited to, sera, plasma, urine, saliva, mucous, pleural fluid, synovial fluid and spinal fluid.
Methods of Ameliorating or Reducing Symptoms of, or Treating, or Preventing, Diseases
and Disorders Associated with, CGRP
In another embodiment described herein, anti-CGRP antibodies described
herein, or fragments thereof, are useful for ameliorating or reducing the symptoms of, or
treating, or preventing, diseases and disorders associated with CGRP. Anti-CGRP
antibodies described herein, or fragments thereof, as well as combinations, can also be
administered in a therapeutically effective amount to patients in need of treatment of
diseases and disorders associated with CGRP in the form of a pharmaceutical composition
as described in greater detail below.
In another embodiment described herein, anti-CGRP antibodies, or fragments
thereof, are useful for ameliorating or reducing the symptoms of, or treating, or preventing,
migraines (with or without aura), weight loss, cancer or tumors, angiogenesis associated
with cancer or tumor growth, angiogenesis associated with cancer or tumor survival, pain,
hemiplagic migraines, cluster headaches, migrainous neuralgia, chronic headaches, tension
headaches, general headaches, hot flushes, chronic paroxysomal hemicrania, secondary
headaches due to an underlying structural problem in the head or neck, cranial neuralgia,
sinus headaches (such as for example associated with sinusitis), and allergy-induced
headaches or migraines.
In one embodiment, anti-CGRP antibodies described herein, or fragments
thereof and/or with a second agent, are useful for ameliorating or reducing the symptoms
of, or treating, or preventing, the following non-limiting listing of diseases and disorders:
pain, inflammatory pain, post-operative incision pain, complex regional pain syndrome,
cancer pain, primary or metastatic bone cancer pain, fracture pain, chronic pain,
osteoporotic fracture pain, pain resulting from burn, osteoporosis, gout joint pain,
abdominal pain, pain associated with sickle cell crises, and other nociceptic pain, as well as
hepatocellular carcinoma, breast cancer, liver cirrhosis, neurogenic pain, neuropathic pain,
nociceptic pain, trigeminal neuralgia, post-herpetic neuralgia, phantom limb pain,
fibromyalgia, menstrual pain, ovarialgia, reflex sympathetic dystrophy, neurogenic pain,
osteoarthritis or rheumatoid arthritis pain, lower back pain, diabetic neuropathy, sciatica, or
pain or visceral pain associated with: gastro-esophageal reflux, dyspepsia, irritable bowel
syndrome, irritable colon, spastic colon, mucous colitis, inflammatory bowel disease,
Crohn’s disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstrual
period, labor, menopause, prostatitis, pancreatitis, renal colic, dysmenorrhea, cystitis,
including interstitial cystitis (IC), surgery associated with the ileus, diverticulitis,
peritonitis, pericarditis, hepatitis, appendicitis, colitis, cholecystitis, endometriosis, chronic
and/or acute pancreatitis, myocardial infarction, kidney pain, pleural pain, prostatitis,
pelvic pain, trauma to an organ, chronic nociceptive pain, chronic neuropathic pain, chronic
inflammatory pain, fibromyalgia, breakthrough pain and persistent pain, and cancer pain
arising from malignancy or from cancer preferably selected from one or more of:
adenocarcinoma in glandular tissue, blastoma in embryonic tissue of organs, carcinoma in
epithelial tissue, leukemia in tissues that form blood cells, lymphoma in lymphatic tissue,
myeloma in bone marrow, sarcoma in connective or supportive tissue, adrenal cancer,
AIDS-related lymphoma, anemia, bladder cancer, bone cancer, brain cancer, breast cancer,
carcinoid tumours, cervical cancer, chemotherapy, colon cancer, cytopenia, endometrial
cancer, esophageal cancer, gastric cancer, head cancer, neck cancer, hepatobiliary cancer,
kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's disease,
lymphoma, non- Hodgkin's, nervous system tumours, oral cancer, ovarian cancer,
pancreatic cancer, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular
cancer, thyroid cancer, urethral cancer, bone cancer, sarcomas cancer of the connective
tissue, cancer of bone tissue, cancer of blood-forming cells, cancer of bone marrow,
multiple myeloma, leukaemia, primary or secondary bone cancer, tumours that metastasize
to the bone, tumours infiltrating the nerve and hollow viscus, tumours near neural
structures. Further preferably the cancer pain comprises visceral pain, preferably visceral
pain which arises from pancreatic cancer and/or metastases in the abdomen. Further
preferably the cancer pain comprises somatic pain, preferably somatic pain due to one or
more of bone cancer, metastasis in the bone, postsurgical pain, sarcomas cancer of the
connective tissue, cancer of bone tissue, cancer of blood-forming cells of the bone marrow,
multiple myeloma, leukaemia, primary or secondary bone cancer.
In another embodiment, anti-CGRP antibodies described herein, or fragments
thereof and/or with a second agent, are useful for ameliorating or reducing the symptoms
of, or treating, or preventing, the following non-limiting listing of diseases and disorders:
cancer or tumors, angiogenesis associated with cancer or tumor growth, angiogenesis
associated with cancer or tumor survival.
In another embodiment, anti-CGRP antibodies described herein, or fragments
thereof and/or with a second agent, are useful for ameliorating or reducing the symptoms
of, or treating, or preventing, the following non-limiting listing of diseases and disorders:
neurogenic, neuropathic or nociceptic pain. Neuropathic pain may include, but is not
limited to, trigeminal neuralgia, post-herpetic neuralgia, phantom limb pain, fibromyalgia,
menstrual pain, ovarialgia, reflex sympathetic dystrophy and neurogenic pain. In other
preferred embodiments, osteoarthritis or rheumatoid arthritis pain, lower back pain,
diabetic neuropathy, sciatica, and other neuropathic pain.
In another embodiment, anti-CGRP antibodies described herein, or fragments
thereof and/or with a second agent, are useful for ameliorating or reducing the symptoms
of, or treating, or preventing, the following non-limiting listing of diseases and disorders:
overactive bladder and other urinary conditions, gastro-esophageal reflux and visceral pain
associated with gastro-esophageal reflux, dyspepsia, irritable bowel syndrome,
inflammatory bowel disease, Crohn’s disease, ileitis, ulcerative colitis, renal colic,
dysmenorrhea, cystitis, menstrual period, labor, menopause, prostatitis, pruritis, or
pancreatitis. Also, the subject CGRP antibodies and antibody fragments may be used alone
or in conjunction with other active agents, e.g., opioids and non-opioid analgesics such as
NSAIDs to elicit analgesia or to potentiate the efficacy of another analgesic or to prevent or
alleviate tolerance to a specific analgesic such as morphine or related opioid analgesics.
Evidence for role of CGRP in blocking / reversing development of morphine – induced
analgesia: is the fact that CGRP8-37 and CGRP Receptor antagonist (BIBN4096BS)
reportedly prevent / reverse development of morphine tolerance -(Powell et al., 2000 J Brit
J Pharmacol (131):875; Menard et al., 1996 J Neurosci (16):2342; Wang et al., 2009
FASEB J (23):2576; Wang et al., 2010 Pain (151):194)
The subject antibodies potentially may be combined with any opioid analgesic
or NSAID or other analgesic, potentially another antibody, in order to increase or enhance
pain management, or to reverse or suppress tolerance to an analgesic such as an opioid
analgesic compound. This may allow for such analgesic compounds to be administered for
longer duration or at reduced dosages thereby potentially alleviating adverse side effects
associated therewith.
The term "opioid analgesic" herein refers to all drugs, natural or synthetic, with
morphine-like actions. The synthetic and semi-synthetic opioid analgesics are derivatives
of five chemical classes of compound: phenanthrenes; phenylheptylamines;
phenylpiperidines; morphinans; and benzomorphans, all of which are within the scope of
the term. Exemplary opioid analgesics include codeine, dihydrocodeine, diacetylmorphine,
hydrocodone, hydromorphone, levorphanol, oxymorphone, alfentanil, buprenorphine,
butorphanol, fentanyl, sufentanyl, meperidine, methadone, nalbuphine, propoxyphene and
pentazocine or pharmaceutically acceptable salts thereof.
The term "NSAID" refers to a non-steroidal anti-inflammatory compound.
NSAIDs are categorized by virtue of their ability to inhibit cyclooxygenase.
Cyclooxygenase 1 and cyclooxygenase 2 are two major isoforms of cyclooxygenase and
most standard NSAIDs are mixed inhibitors of the two isoforms. Most standard NSAIDs
fall within one of the following five structural categories: (1) propionic acid derivatives,
such as ibuprofen, naproxen, naprosyn, diclofenac, and ketoprofen; (2) acetic acid
derivatives, such as tolmetin and slindac; (3) fenamic acid derivatives, such as mefenamic
acid and meclofenamic acid; (4) biphenylcarboxylic acid derivatives, such as diflunisal and
flufenisal; and (5) oxicams, such as piroxim, sudoxicam, and isoxicam. Another class of
NSAID has been described which selectively inhibit cyclooxygenase 2. Cox-2 inhibitors
have been described, e.g., in U.S. Pat. Nos. 5,616,601; 5,604,260; 5,593,994; 5,550,142;
,536,752; 5,521,213; 5,475,995; 5,639,780; 5,604,253; 5,552,422; 5,510,368; 5,436,265;
,409,944; and 5,130,311, all of which are hereby incorporated by reference. Certain
exemplary COX-2 inhibitors include celecoxib (SC-58635), DUP-697, flosulide (CGP-
28238), meloxicam, 6-methoxy-2 naphthylacetic acid (6-MNA), rofecoxib, MK-966,
nabumetone (prodrug for 6-MNA), nimesulide, NS-398, SC-5766, SC-58215, T-614; or
combinations thereof.
In some embodiments, aspirin and/or acetaminophen may be taken in
conjunction with the subject CGRP antibody or fragment. Aspirin is another type of non-
steroidal anti-inflammatory compound.
Exemplary, non-limiting diseases and disorders that can be treated and/or
prevented by the administration of the CGRP antibodies of the present disclosure include,
pain resulting from any condition associated with neurogenic, neuropathic, inflammatory,
thermal or nociceptic pain. Preferably the disorder will be associated with increased CGRP
at the pain site. In certain embodiments of neuropathic pain, referred trigeminal neuralgia,
post-herpetic neuralgia, phantom limb pain, fibromyalgia, reflex sympathetic dystrophy
and neurogenic pain conditions are preferably treated. In other embodiments, cancer pain,
particularly, bone cancer pain, osteoarthritis or rheumatoid arthritis pain, lower back pain,
post-operative incision pain, fracture pain, osteoporotic fracture pain, osteoporosis, gout
joint pain, diabetic neuropathy, sciatica, pains associated with sickle cell crises, migraine,
and other neuropathic and/or nociceptic pain are preferably treated. Thus, the present
discosure includes methods of treating, preventing, and/or ameliorating any disease or
disorder associated with CGRP activity or CGRP upregulation (including any of the above
mentioned exemplary diseases, disorders and conditions) through use of the antibodies and
antibody fragments described herein. The therapeutic methods described herein comprise
administering to a subject any formulation comprising an anti-CGRP antibody as disclosed
herein alone or in association with another active agent.
. The subject to which the pharmaceutical formulation is administered can be,
e.g., any human or non-human animal that is in need of such treatment, prevention and/or
amelioration, or who would otherwise benefit from the inhibition or attenuation of CGRP-
mediated activity. For example, the subject can be an individual that is diagnosed with, or
who is deemed to be at risk of being afflicted by any of the aforementioned diseases or
disorders. The present disclosure further includes the use of any of the pharmaceutical
formulations disclosed herein in the manufacture of a medicament for the treatment,
prevention and/or amelioration of any disease or disorder associated with CGRP activity
(including any of the above mentioned exemplary diseases, disorders and conditions).
Administration
In one embodiment, the anti-CGRP antibodies described herein, or CGRP
binding fragments thereof, as well as combinations of said antibodies or antibody
fragments, are administered to a subject at a concentration of between about 0.1 and 100.0
mg/kg of body weight of recipient subject. In a preferred embodiment, the anti-CGRP
antibodies described herein, or CGRP binding fragments thereof, as well as combinations
of said antibodies or antibody fragments, are administered to a subject at a concentration of
about 0.4 mg/kg of body weight of recipient subject. In a preferred embodiment, the anti-
CGRP antibodies described herein, or CGRP binding fragments thereof, as well as
combinations of said antibodies or antibody fragments, are administered to a recipient
subject with a frequency of once every twenty-six weeks or less, such as once every sixteen
weeks or less, once every eight weeks or less, once every four weeks or less, once every
two weeks or less, once every week or less, or once daily or less.
Fab fragments may be administered every two weeks or less, every week or less,
once daily or less, multiple times per day, and/or every few hours. In one embodiment
described herein, a patient receives Fab fragments of 0.1 mg/kg to 40 mg/kg per day given
in divided doses of 1 to 6 times a day, or in a sustained release form, effective to obtain
desired results.
It is to be understood that the concentration of the antibody or Fab administered
to a given patient may be greater or lower than the exemplary administration
concentrations set forth above in paragraphs [0552] and [0553].
A person of skill in the art would be able to determine an effective dosage and
frequency of administration through routine experimentation, for example guided by the
disclosure herein and the teachings in Goodman, L. S., Gilman, A., Brunton, L. L., Lazo, J.
S., & Parker, K. L. (2006). Goodman & Gilman's the pharmacological basis of
therapeutics. New York: McGraw-Hill; Howland, R. D., Mycek, M. J., Harvey, R. A.,
Champe, P. C., & Mycek, M. J. (2006). Pharmacology. Lippincott's illustrated reviews.
Philadelphia: Lippincott Williams & Wilkins; and Golan, D. E. (2008). Principles of
pharmacology: the pathophysiologic basis of drug therapy. Philadelphia, Pa., [etc.]:
Lippincott Williams & Wilkins.
In another embodiment, the anti-CGRP antibodies described herein, or CGRP
binding fragments thereof, as well as combinations of said antibodies or antibody
fragments, are administered to a subject in a pharmaceutical formulation.
A “pharmaceutical composition” refers to a chemical or biological composition
suitable for administration to a mammal. Such compositions may be specifically
formulated for administration via one or more of a number of routes, including but not
limited to buccal, epicutaneous, epidural, inhalation, intraarterial, intracardial,
intracerebroventricular, intradermal, intramuscular, intranasal, intraocular, intraperitoneal,
intraspinal, intrathecal, intravenous, oral, parenteral, rectally via an enema or suppository,
subcutaneous, subdermal, sublingual, transdermal, and transmucosal. In addition,
administration can occur by means of injection, powder, liquid, gel, drops, or other means
of administration.
In one embodiment, the anti-CGRP antibodies described herein, or CGRP
binding fragments thereof, as well as combinations of said antibodies or antibody
fragments, may be optionally administered in combination with one or more active agents.
Such active agents include analgesic, anti-histamine, antipyretic, anti-inflammatory,
antibiotic, antiviral, and anti-cytokine agents. Active agents include agonists, antagonists,
and modulators of TNF-α, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-α, IFN-γ,
BAFF, CXCL13, IP-10, VEGF, EPO, EGF, HRG, Hepatocyte Growth Factor (HGF),
Hepcidin, including antibodies reactive against any of the foregoing, and antibodies
reactive against any of their receptors. Active agents also include but are not limited to 2-
Arylpropionic acids, Aceclofenac, Acemetacin, Acetylsalicylic acid (Aspirin), Alclofenac,
Alminoprofen, Amoxiprin, Ampyrone, Arylalkanoic acids, Azapropazone,
Benorylate/Benorilate, Benoxaprofen, Bromfenac, Carprofen, Celecoxib, Choline
magnesium salicylate, Clofezone, COX-2 inhibitors, Dexibuprofen, Dexketoprofen,
Diclofenac, Diflunisal, Droxicam, Ethenzamide, Etodolac, Etoricoxib, Faislamine, fenamic
acids, Fenbufen, Fenoprofen, Flufenamic acid, Flunoxaprofen, Flurbiprofen, Ibuprofen,
Ibuproxam, Indometacin, Indoprofen, Kebuzone, Ketoprofen, Ketorolac, Lornoxicam,
Loxoprofen, Lumiracoxib, Magnesium salicylate, Meclofenamic acid, Mefenamic acid,
Meloxicam, Metamizole, Methyl salicylate, Mofebutazone, Nabumetone, Naproxen, N-
Arylanthranilic acids, Nerve Growth Factor (NGF), Oxametacin, Oxaprozin, Oxicams,
Oxyphenbutazone, Parecoxib, Phenazone, Phenylbutazone, Phenylbutazone, Piroxicam,
Pirprofen, profens, Proglumetacin, Pyrazolidine derivatives, Rofecoxib, Salicyl salicylate,
Salicylamide, Salicylates, Substance P, Sulfinpyrazone, Sulindac, Suprofen, Tenoxicam,
Tiaprofenic acid, Tolfenamic acid, Tolmetin, and Valdecoxib.
An anti-histamine can be any compound that opposes the action of histamine or
its release from cells (e.g., mast cells). Anti-histamines include but are not limited to
acrivastine, astemizole, azatadine, azelastine, betatastine, brompheniramine, buclizine,
cetirizine, cetirizine analogues, chlorpheniramine, clemastine, CS 560, cyproheptadine,
desloratadine, dexchlorpheniramine, ebastine, epinastine, fexofenadine, HSR 609,
hydroxyzine, levocabastine, loratidine, methscopolamine, mizolastine, norastemizole,
phenindamine, promethazine, pyrilamine, terfenadine, and tranilast.
Antibiotics include but are not limited to Amikacin, Aminoglycosides,
Amoxicillin, Ampicillin, Ansamycins, Arsphenamine, Azithromycin, Azlocillin,
Aztreonam, Bacitracin, Carbacephem, Carbapenems, Carbenicillin, Cefaclor, Cefadroxil,
Cefalexin, Cefalothin, Cefalotin, Cefamandole, Cefazolin, Cefdinir, Cefditoren, Cefepime,
Cefixime, Cefoperazone, Cefotaxime, Cefoxitin, Cefpodoxime, Cefprozil, Ceftazidime,
Ceftibuten, Ceftizoxime, Ceftobiprole, Ceftriaxone, Cefuroxime, Cephalosporins,
Chloramphenicol, Cilastatin, Ciprofloxacin, Clarithromycin, Clindamycin, Cloxacillin,
Colistin, Co-trimoxazole, Dalfopristin, Demeclocycline, Dicloxacillin, Dirithromycin,
Doripenem, Doxycycline, Enoxacin, Ertapenem, Erythromycin, Ethambutol,
Flucloxacillin, Fosfomycin, Furazolidone, Fusidic acid, Gatifloxacin, Geldanamycin,
Gentamicin, Glycopeptides, Herbimycin, Imipenem, Isoniazid, Kanamycin, Levofloxacin,
Lincomycin, Linezolid, Lomefloxacin, Loracarbef, Macrolides, Mafenide, Meropenem,
Meticillin, Metronidazole, Mezlocillin, Minocycline, Monobactams, Moxifloxacin,
Mupirocin, Nafcillin, Neomycin, Netilmicin, Nitrofurantoin, Norfloxacin, Ofloxacin,
Oxacillin, Oxytetracycline, Paromomycin, Penicillin, Penicillins, Piperacillin,
Platensimycin, Polymyxin B, Polypeptides, Prontosil, Pyrazinamide, Quinolones,
Quinupristin, Rifampicin, Rifampin, Roxithromycin, Spectinomycin, Streptomycin,
Sulfacetamide, Sulfamethizole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Sulfonamides,
Teicoplanin, Telithromycin, Tetracycline, Tetracyclines, Ticarcillin, Tinidazole,
Tobramycin, Trimethoprim, Trimethoprim-Sulfamethoxazole, Troleandomycin,
Trovafloxacin, and Vancomycin.
Active agents also include Aldosterone, Beclometasone, Betamethasone,
Corticosteroids, Cortisol, Cortisone acetate, Deoxycorticosterone acetate, Dexamethasone,
Fludrocortisone acetate, Glucocorticoids, Hydrocortisone, Methylprednisolone,
Prednisolone, Prednisone, Steroids, and Triamcinolone. Any suitable combination of these
active agents is also contemplated.
A “pharmaceutical excipient” or a “pharmaceutically acceptable excipient” is a
carrier, usually a liquid, in which an active therapeutic agent is formulated. In one
embodiment described herein, the active therapeutic agent is a humanized antibody
described herein, or one or more fragments thereof. The excipient generally does not
provide any pharmacological activity to the formulation, though it may provide chemical
and/or biological stability, and release characteristics. Exemplary formulations can be
found, for example, in Remington’s Pharmaceutical Sciences, 19 Ed., Grennaro, A., Ed.,
1995 which is incorporated by reference.
As used herein “pharmaceutically acceptable carrier” or “excipient” includes any
and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic
and absorption delaying agents that are physiologically compatible. In one embodiment,
the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable
for intravenous, intraperitoneal, intramuscular, or sublingual administration.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile injectable solutions or
dispersions. The use of such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or agent is incompatible
with the active compound, use thereof in the pharmaceutical compositions described herein
is contemplated. Supplementary active compounds can also be incorporated into the
compositions.
Pharmaceutical compositions typically must be sterile and stable under the
conditions of manufacture and storage. The present disclosure contemplates that the
pharmaceutical composition is present in lyophilized form. The composition can be
formulated as a solution, microemulsion, liposome, or other ordered structure suitable to
high drug concentration. The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol), and suitable mixtures thereof. The present disclosure further
contemplates the inclusion of a stabilizer in the pharmaceutical composition. The proper
fluidity can be maintained, for example, by the maintenance of the required particle size in
the case of dispersion and by the use of surfactants.
In many cases, it will be preferable to include isotonic agents, for example,
sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought about by including in
the composition an agent which delays absorption, for example, monostearate salts and
gelatin. Moreover, the alkaline polypeptide can be formulated in a time release
formulation, for example in a composition which includes a slow release polymer. The
active compounds can be prepared with carriers that will protect the compound against
rapid release, such as a controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used,
such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many
methods for the preparation of such formulations are known to those skilled in the art.
For each of the recited embodiments, the compounds can be administered by a
variety of dosage forms. Any biologically-acceptable dosage form known to persons of
ordinary skill in the art, and combinations thereof, are contemplated. Examples of such
dosage forms include, without limitation, reconstitutable powders, elixirs, liquids,
solutions, suspensions, emulsions, powders, granules, particles, microparticles, dispersible
granules, cachets, inhalants, aerosol inhalants, patches, particle inhalants, implants, depot
implants, injectables (including subcutaneous, intramuscular, intravenous, and
intradermal), infusions, and combinations thereof.
The above description of various illustrated embodiments of the invention is not
intended to be exhaustive or to limit the invention to the precise form disclosed. While
specific embodiments of, and examples for, the invention are described herein for
illustrative purposes, various equivalent modifications are possible within the scope of the
invention, as those skilled in the relevant art will recognize. The teachings provided herein
of the invention can be applied to other purposes, other than the examples described above.
These and other changes can be made to the invention in light of the above
detailed description. In general, in the following claims, the terms used should not be
construed to limit the invention to the specific embodiments disclosed in the specification
and the claims. Accordingly, the invention is not limited by the disclosure, but instead the
scope of the invention is to be determined entirely by the following claims.
The invention may be practiced in ways other than those particularly described
in the foregoing description and examples. Numerous modifications and variations of the
invention are possible in light of the above teachings and, therefore, are within the scope of
the appended claims.
Certain teachings related to methods for obtaining a clonal population of
antigen-specific B cells were disclosed in U.S. Provisional patent application no.
60/801,412, filed May 19, 2006, the disclosure of which is herein incorporated by reference
in its entirety.
Certain teachings related to humanization of rabbit-derived monoclonal
antibodies and preferred sequence modifications to maintain antigen binding affinity were
disclosed in International Application No. , corresponding to
International Publication No. WO/2008/144757, entitled “Novel Rabbit Antibody
Humanization Methods and Humanized Rabbit Antibodies”, filed May 21, 2008, the
disclosure of which is herein incorporated by reference in its entirety.
Certain teachings related to producing antibodies or fragments thereof using
mating competent yeast and corresponding methods were disclosed in U.S. Patent
application no. 11/429,053, filed May 8, 2006, (U.S. Patent Application Publication No.
US2006/0270045), the disclosure of which is herein incorporated by reference in its
entirety.
Certain CGRP antibody polynucleotides and polypeptides are disclosed in the
sequence listing accompanying this patent application filing, and the disclosure of said
sequence listing is herein incorporated by reference in its entirety.
The entire disclosure of each document cited (including patents, patent
applications, journal articles, abstracts, manuals, books, or other disclosures) in the
Background of the Invention, Detailed Description, and Examples is herein incorporated by
reference in their entireties.
The following examples are put forth so as to provide those of ordinary skill in
the art with a complete disclosure and description of how to make and use the subject
invention, and are not intended to limit the scope of what is regarded as the invention.
Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts,
temperature, concentrations, etc.) but some experimental errors and deviations should be
allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is
average molecular weight, temperature is in degrees centigrade; and pressure is at or near
atmospheric.
EXAMPLES
Example 1 Preparation of Antibodies that Bind CGRP
By using the antibody selection protocol described herein, one can generate an
extensive panel of antibodies.
Immunization Strategy
Rabbits were immunized with human CGRPα (American Peptides, Sunnyvale
CA and Bachem, Torrance CA). Immunization consisted of a first subcutaneous (sc)
injection of 100 μg of antigen mixed with 100 μg of KLH in complete Freund’s adjuvant
(CFA) (Sigma) followed by two boosts, two weeks apart each containing 50 μg antigen
mixed with 50 μg in incomplete Freund’s adjuvant (IFA) (Sigma). Animals were bled on
day 55, and serum titers were determined by ELISA (antigen recognition) and by inhibition
of CGRP driven cAMP increase in SK-N-MC.
Antibody Selection Titer Assessment
To identify and characterize antibodies that bind to human CGRPα, antibody-
containing solutions were tested by ELISA. Briefly, neutravidin coated plates (Thermo
Scientific), were coated with N-term biotinylated human CGRPα (50μL per well, 1μg/mL)
diluted in ELISA buffer (0.5% fish skin gelatin in PBS pH 7.4,) either for approximately
1hr at room temperature or alternatively overnight at 4°C. The plates were then further
blocked with ELISA buffer for one hour at room temperature and washed using wash
buffer (PBS, 0.05% tween 20). Serum samples tested were serially diluted using ELISA
buffer. Fifty microliters of diluted serum samples were transferred onto the wells and
incubated for one hour at room temperature for one hour. After this incubation, the plate
was washed with wash buffer. For development, an anti-rabbit specific Fc-HARP (1:5000
dilution in ELISA buffer) was added onto the wells and incubated for 45 min at RT. After
a 3x wash step with wash solution, the plate was developed using TMB substrate for two
minutes at room temperature and the reaction was quenched using 0.5M HCl. The well
absorbance was read at 450 nm.
Titer determination of serum samples by functional activity (Inhibition of CGRP driven
cAMP levels)
To identify and characterize antibodies with functional activity, an inhibition of
CGRP driven increase of cAMP levels assay was done using electrochemiluminescence
(Meso Scale Discovery, MSD). Briefly, antibody preparations to be tested were serially
diluted in MSD assay buffer (Hepes, MgCl2, pH 7.3, 1mg/mL blocker A, Meso Scale
Discovery) in a 96 well round bottom polystyrene plate (Costar). To this plate, human
CGRPα was added (10ng/mL final concentration) diluted in MSD assay buffer and
incubated for one hour at 37C. Appropriate controls were used as suggested by the assay-
kit manufacturer. Human neuroepithelioma cells (SK-N-MC, ATCC) were detached using
an EDTA solution (5mM in PBS) and washed using growth media (MEM, 10% FBS,
antibiotics) by centrifugation. The cell number was adjusted to 2 million cells per mL in
assay buffer, and IBMX (3-Isobutyl-1Methylxanthine, Sigma) was added to a final
concentration of 0.2mM right before loading cells onto cAMP assay plate. After the
antibody human CGRPα solution was incubated for one hour 20 microliters of solution
containing cells were transferred to the cAMP assay plate. All tested samples were run in
duplicates with appropriate controls. Ten microliters of cells were added to the wells and
the plate was incubated for 30 minutes with shaking at room temperature. While cells were
being incubated with the CGRP solution, the stop solution was prepared by making a 1:200
solution of TAG labeled cAMP (MSD) in lysis buffer (MSD). To stop the cells-CGRP
incubation, 20 microliters of stop solution was added to the cells and the plate was
incubated for one hour with shaking at room temperature. The read buffer (MSD) was
diluted four times with water and 100 microliters were added to all wells on the plate. The
plate was then read using a Sector Imager 2400 (MSD) and the Prism software was used
for data fit and IC50 determination.
Tissue Harvesting
Once acceptable titers were established, the rabbit(s) were sacrificed. Spleen,
lymph nodes, and whole blood were harvested and processed as follows:
Spleen and lymph nodes were processed into a single cell suspension by
disassociating the tissue and pushing through sterile wire mesh at 70 μm (Fisher) with a
plunger of a 20 cc syringe. Cells were collected in PBS. Cells were washed twice by
centrifugation. After the last wash, cell density was determined by trypan blue. Cells were
centrifuged at 1500 rpm for 10 minutes; the supernatant was discarded. Cells were
resuspended in the appropriate volume of 10% dimethyl sulfoxide (DMSO, Sigma) in FBS
(Hyclone) and dispensed at 1 ml/vial. Vials were stored at -70°C in a slow freezing
chamber for 24 hours and stored in liquid nitrogen.
Peripheral blood mononuclear cells (PBMCs) were isolated by mixing whole
blood with equal parts of the low glucose medium described above without FBS. 35 ml of
the whole blood mixture was carefully layered onto 8 ml of Lympholyte Rabbit
(Cedarlane) into a 45 ml conical tube (Corning) and centrifuged 30 minutes at 2500 rpm at
room temperature without brakes. After centrifugation, the PBMC layers were carefully
removed using a glass Pasteur pipette (VWR), combined, and placed into a clean 50 ml
vial. Cells were washed twice with the modified medium described above by
centrifugation at 1500 rpm for 10 minutes at room temperature, and cell density was
determined by trypan blue staining. After the last wash, cells were resuspended in an
appropriate volume of 10% DMSO/FBS medium and frozen as described above.
B cell selection, enrichment and culture conditions
On the day of setting up B cell culture, PBMC, splenocyte, or lymph node vials
were thawed for use. Vials were removed from LN2 tank and placed in a 37°C water bath
until thawed. Contents of vials were transferred into 15 ml conical centrifuge tube
(Corning) and 10 ml of modified RPMI described above was slowly added to the tube.
Cells were centrifuged for 5 minutes at 2K RPM, and the supernatant was discarded. Cells
were resuspended in 10 ml of fresh media. Cell density and viability was determined by
trypan blue.
a) The following protocol was used for Ab1 and Ab13
Cells were pre-mixed with the biotinylated human CGRPα as follows. Cells
were washed again and resuspended at 1E07 cells/80 μL medium. Biotinylated human
CGRPα was added to the cell suspension at the final concentration of 5 ug/mL and
incubated for 30 minutes at 4°C. Unbound biotinylated human CGRPα was removed
performing two 10 ml washes using PBF [Ca/Mg free PBS (Hyclone), 2 mM
ethylenediamine tetraacetic acid (EDTA), 0.5% bovine serum albumin (BSA) (Sigma-
biotin free)]. After the second wash, cells were resuspended at 1E07 cells/80 μl PBF and
μl of MACS® streptavidin beads (Miltenyi Biotech, Auburn CA) per 10E7 cells were
added to the cell suspension. Cells and beads were incubated at 4°C for 15 minutes and
washed once with 2 ml of PBF per 10E7 cells.
b) The following protocol was used for Ab4, Ab7, Ab9 and Ab11:
Biotinylated human CGRPα was pre-loaded onto the streptavidin beads as
follows. Seventy five microliters of streptavidin beads (Milteny Biotec, Auburn CA) were
mixed with N-terminally biotinylated huCGRPα (10ug/ml final concentration) and 300 μl
PBF. This mixture was incubated at 4°C for 30 min and unbound biotinylated human
CGRPα was removed using a MACS® separation column (Miltenyi Biotec, with a 1ml
rinse to remove unbound material. Then material was plunged out, then used to resuspend
cells from above in 100ul per 1E7 cells, the mixture was then incubated at 4°C for 30min
and washed once with 10 ml of PBF.
For both a) and b) protocols the following applied: After washing, the cells were
resuspended in 500μl of PBF and set aside. A MACS® MS column (Miltenyi Biotec,
Auburn CA) was pre-rinsed with 500 ml of PBF on a magnetic stand (Milteni). Cell
suspension was applied to the column through a pre-filter, and unbound fraction was
collected. The column was washed with 2.5 ml of PBF buffer. The column was removed
from the magnet stand and placed onto a clean, sterile 1.5 ml eppendorf tube. 1 ml of PBF
buffer was added to the top of the column, and positive selected cells were collected. The
yield and viability of positive cell fraction was determined by trypan blue staining. Positive
selection yielded an average of 1% of the starting cell concentration.
A pilot cell screen was established to provide information on seeding levels for
the culture. Plates were seeded at 10, 25, 50, 100, or 200 enriched B cells/well. In
addition, each well contained 50K cells/well of irradiated EL-4.B5 cells (5,000 Rads) and
an appropriate level of activated rabbit T cell supernatant (See U.S. Patent Application
Publication No. 20070269868)(ranging from 1-5% depending on preparation) in high
glucose modified RPMI medium at a final volume of 250 μl/well. Cultures were incubated
for 5 to 7 days at 37°C in 4% CO .
B-Cell culture screening by antigen-recognition (ELISA)
To identify wells producing anti-human CGRPα antibodies, the same protocol as
described for titer determination of serum samples by antigen-recognition (ELISA) was
used with the following changes. Briefly, neutravidin coated plates were coated with a
mixture of both N- and C- terminally biotinylated human CGRPα (50μL per well, 1μg/mL
each). B-cell supernatant samples (50μL) were tested without prior dilution.
Identification of functional activity in B-cell supernatants using CGRP driven cAMP
production
To determine functional activity contained in B-cell supernatants, a similar
procedure to that described for the determination of functional titer of serum samples was
used with the following modifications. Briefly, B-cell supernatant (20μL) were used in
place of the diluted polyclonal serum samples.
Isolation of antigen-specific B-cells
Plates containing wells of interest were removed from -70 °C, and the cells from
each well were recovered using five washes of 200 microliters of medium (10% RPMI
complete, 55μM BME) per well. The recovered cells were pelleted by centrifugation and
the supernatant was carefully removed. Pelleted cells were resuspended in 100 μl of
medium. To identify antibody expressing cells, streptavidin coated magnetic beads (M280
dynabeads, Invitrogen) were coated with a combination of both N- and C- terminal
biotinylated human CGRPα. Individual biotinylated human CGRPα lots were optimized
by serial dilution. One hundred microliters containing approximately 4x10E7 coated beads
were then mixed with the resuspended cells. To this mixture 15 microliters of goat anti-
rabbit H&L IgG-FITC (Jackson Immunoresearch) diluted 1:100 in medium were added.
Twenty microliters of cell/beads/anti-rabbit H&L suspension were removed and
microliter droplets were dispensed on a one-well glass slide previously treated with
Sigmacote (Sigma) totaling 35 to 40 droplets per slide. An impermeable barrier of paraffin
oil (JT Baker) was used to submerge the droplets, and the slide was incubated for 90
minutes at 37°C in a 4% CO2 incubator in the dark.
Specific B cells that produce antibody can be identified by the fluorescent ring
around produced by the antibody secretion, recognition of the bead-associated biotinylated
antigen, and subsequent detection by the fluorescent-IgG detection reagent. Once a cell of
interest was identified it was recovered via a micromanipulator (Eppendorf). The single
cell synthesizing and exporting the antibody was transferred into a microcentrifuge tube,
frozen using dry ice and stored at -70°C.
Amplification and sequence determination of Antibody Sequences From Antigen-Specific
B Cells
Antibody sequences were recovered using a combined RT-PCR based method
from a single isolated B-cell. Primers containing restriction enzymes were designed to
anneal in conserved and constant regions of the target immunoglobulin genes (heavy and
light), such as rabbit immunoglobulin sequences, and a two-step nested PCR recovery was
used to amplify the antibody sequence. Amplicons from each well were analyzed for
recovery and size integrity. The resulting fragments are then digested with AluI to
fingerprint the sequence clonality. Identical sequences displayed a common fragmentation
pattern in their electrophoretic analysis. The original heavy and light chain amplicon
fragments were then digested using the restriction enzyme sites contained within the PCR
primers and cloned into an expression vector. Vector containing subcloned DNA
fragments were amplified and purified. Sequence of the subcloned heavy and light chains
were verified prior to expression.
Recombinant Production of Monoclonal Antibody of Desired Antigen Specificity and/or
Functional Properties
To determine antigen specificity and functional properties of recovered
antibodies from specific B-cells, vectors driving the expression of the desired paired heavy
and light chain sequences were transfected into HEK-293 cells.
Antigen-recognition of recombinant antibodies by ELISA
To characterize recombinant expressed antibodies for their ability to bind to
human-CGRPα, antibody-containing solutions were tested by ELISA. All incubations were
done at room temperature. Briefly, Immulon IV plagtes (Thermo Scientific) were coated
with a CGRPα containing solution (1ut/mL in PBS) for 2 hours. CGRPα-coated plates
were then washed three times in wash buffer (PBS, 0.05% Tween-20). The plates were
then blocked using a blocking solution (PBS, 0.5% fish skin gelatin, 0.05% Tween-20) for
approximately one hour. The blocking solution was then removed and the plates were then
incubated with a dilution series of the antibody being tested for approximately one hour.
At the end of this incubation, the plate was washed three times with wash buffer and
further incubated with a secondary antibody containing solution (Peroxidase conjugated
affinipure F(ab’)2 fragment goat anti-human IgG, Fc fragment specific (Jackson
Immunoresearch) for approximately 45 minutes and washed three times. At that point a
substrate solution (TMB peroxidase substrate, BioFx) and incubated for 3 to 5 minutes in
the dark. The reaction was stopped by addition of a HCl containing solution (0.5M) and
the plate was read at 450 nm in a plate-reader.
Results: Figures 15-18 demonstrate that anti-CGRP antibodies Ab1-Ab14 bind
to and recognize CGRPα.
Functional characterization of recombinant antibodies by modulation of CGRP driven
intracellular cAMP levels and cross reactivity to rats
To characterize recombinant expressed antibody for their ability to inhibit
CGRPα mediated increased cellular levels of cAMP assay, an electrochemiluminescence
assay-kit (Meso Scale Discovery, MSD) was used. Briefly, antibody preparations to be
tested were serially diluted in MSD assay buffer (Hepes, MgCl2, pH 7.3, 1mg/mL blocker
A,Meso Scale Discovery) in a 96 well round bottom polystyrene plate (Costar). To this
plate, human CGRPα was added (25ng/mL final concentration) diluted in MSD assay
buffer and incubated for one hour at 37°C. Appropriate controls were used as suggested by
the assay-kit manufacturer. Human neuroepithelioma cells (SK-N-MC, ATCC) were
detached using an EDTA solution (5mM) and washed using growth media (MEM, 10%
FBS, antibiotics) by centrifugation. The cell number was adjusted to 2 million cells per
mL in assay buffer, and IBMX (3-Isobutyl-1Methylxanthine, 50mM Sigma) was added to a
final concentration of 0.2mM right before loading cells onto cAMP assay plate. The
antibody human CGRPα solution was incubated for one hour after which 20 microliters of
solution containing cells were transferred to the cAMP assay plate. All tested samples
were run in duplicates with appropriate controls. Ten microliters of cells were added to the
wells and the plate was incubated for 30 minutes with shaking. While cells were being
incubated with the CGRP solution, the stop solution was prepared by making a 1:200
solution of TAG labeled cAMP (MSD) in lysis buffer (MSD). To stop the cells-CGRP
incubation, 20 microliters of stop solution was added to the cells and the plate was
incubated for one hour with shaking. The read buffer (MSD) was diluted four times with
water and 100 microliters were added to all wells on the plate. The plate was then read
using a Sector Imager 2400 (MSD) and the Prism software was used for data fit and IC50
determination.
To test for the ability of recombinant antibodies to antagonize human CGRPβ a
similar assay was performed with the substitution of the CGRP agonist (CGRPβ 10ng/mL
final concentration). Evaluation of the recombinant antibodies to recognize and inhibit rat
CGRP-mediated cAMP generation was conducted using rat CGRP (5ng/mL final
concentration) and the rat L6 cell line (ATCC).
Results: Figures 19-37 demonstrate that anti-CGRP antibodies Ab1-Ab14
inhibit CGRPα, CGRPβ, and rat CGRP mediated increased cellular levels of cAMP.
Example 2: Enzymatic Production of Fab Fragments
Papain digestions were conducted using immobilized papain (Thermo/Pierce) as
per manufacturer’s instructions. Briefly, purified antibodies were incubated in a
cystein/HCl-containing buffer with immobilized papain at 37°C with gentle rocking. The
digestion was monitored by taking an aliquot and analyzing using SDS-PAGE for cleavage
of the heavy chain. To stop the reaction, the immobilized papain was spun out and washed
using 50 mM Tris pH 7.5 and filtered. Undigested full length antibody and Fc fragments
were removed by using a MabSelectSure (GE) column.
Example 3 Yeast Cell Expression
Construction of Pichia pastoris expression vectors for heavy and light chain.
The humanized light and heavy chain fragments were commercially synthesized
and subcloned into a pGAP expression vector. The pGAP expression vector uses the GAP
promoter to drive expression of the immunoglobulin chain and the human serum albumin
(HSA) leader sequence for export. In addition, this vector contains common elements such
as a bacterial origin of replication, and a copy of the kanamycin resistance gene which
confers resistance to the antibiotic G418 in P. pastoris. G418 provides a means of
selection for strains that contain the desired expression vector integrated into their genome.
Transformation of expression vectors into haploid met1 and lys3 host strains of Pichia
pastoris
All methods used for transformation of haploid P. pastoris strains and
manipulation of the P. pastoris sexual cycle were done as described in Pichia Protocols
(Methods in Molecular Biology Higgings, DR, and Cregg, JM, Eds. 1998. Humana Press,
Totowa, NJ). Prior to transformation each vector was linearized within the GAP promoter
sequences to direct the integration of the vector into the GAP promoter locus of the P.
pastoris genome. Haploid strains were transfected using electroporation and successful
transformants were selected on YPDS (yeast extract, peptone dextrose with sorbitol) G418
agar plates. Copy numbers of heavy and light chain genes were determined for haploid
strains by Southern blot analysis. Haploid strains were then mated and selected for their
ability to grow in the absence of the amino acid markers (i.e., Lys and Met). Resulting
diploid clones were then subjected to a final Southern blot to confirm copy numbers of
heavy and light chain genes. A clone expressing the antibody of interest was selected using
biolayer interferometry Protein-A biosensors to monitor expression (Octet, ForteBio).
Example 4 Expression of Ab3, Ab6 and Ab14 in Pichia pastoris
Three Pichia strains for expression of full-length antibody were made. For all
the full length antibody expressing strains, haploids strains were created and subsequently
mated. One haploid strain expressed full-length light chain sequence and another haploid
strain expressed the full-length heavy chain sequence. Each diploid strain was used to
generate a research cell bank and used for expression in a bioreactor.
First an inoculum was expanded using the research cell bank using medium
comprised of the following nutrients (%w/v): yeast extract 3%, anhydrous dextrose 4%,
YNB 1.34%, Biotin 0.004% and 100 mM potassium phosphate. To generate the inoculum
for the fermenters, the cell bank was expanded for approximately 24 hours in a shaking
incubator at 30ºC and 300 rpm. A 10% inoculum was then added to Labfors 2.5L working
volume vessels containing 1 L sterile growth medium. The growth medium was comprised
of the following nutrients: potassium sulfate 18.2 g/L, ammonium phosphate monobasic
36.4 g/L, potassium phosphate dibasic 12.8 g/L, magnesium sulfate heptahydrate 3.72 g/L,
sodium citrate dihydrate 10 g/L, glycerol 40 g/L, yeast extract 30 g/L, PTM1 trace metals
4.35 mL/L, and antifoam 204 1.67 mL/L. The PTM1 trace metal solution was comprised
of the following components: cupric sulfate pentahydrate 6 g/L, sodium iodide 0.08 g/L,
manganese sulfate hydrate 3 g/L, sodium molybdate dihyrate 0.2 g/L, boric acid 0.02 g/L,
cobalt chloride 0.5 g/L, zinc chloride 20 g/L, ferrous sulfate heptahydrate 65 g/L, biotin 0.2
g/L, and sulfuric acid 5 mL/L.
The bioreactor process control parameters were set as follows: Agitation 1000
rpm, airflow 1.35 standard liter per minute, temperature 28ºC and pH was controlled at six
using ammonium hydroxide. No oxygen supplementation was provided.
Fermentation cultures were grown for approximately 12 to 16 hours until the
initial glycerol was consumed as denoted by a dissolved oxygen spike. The cultures were
starved for approximately three hours after the dissolved oxygen spike. After this
starvation period, a bolus addition of ethanol was added to the reactor to reach 1% ethanol
(w/v). The fermentation cultures were allowed to equilibrate for 15 to 30 minutes. Feed
addition was initiated 30 minutes post-ethanol bolus and set at a constant rate of 1 mL/min
for 40 minutes, then the feed pump was controlled by an ethanol sensor keeping the
concentration of ethanol at 1% for the remainder of the run using an ethanol sensing probe
(Raven Biotech). The feed was comprised of the following components: yeast extract 50
g/L, dextrose 500 g/L, magnesium sulfate heptahydrate 3 g/L, and PTM1 trace metals 12
mL/L. For fermentation of the full length Ab6 and Ab14, sodium citrate dihydrate (0.5g/L)
was also added to the feed. The total fermentation time was approximately 90 hours.
Example 5 Methods of Humanizing Antibodies
Methods of humanizing antibodies have been described previously in issued
U.S. Patent No. 7935340, the disclosure of which is incorporated herein by reference in its
entirety. In some instances, a determination of whether additional rabbit framework
residues are required to maintain activity is necessary. In some instances the humanized
antibodies still requires some critical rabbit framework residues to be retained to minimize
loss of affinity or activity. In these cases, it is necessary to change single or multiple
framework amino acids from human germline sequences back to the original rabbit amino
acids in order to have desired activity. These changes are determined experimentally to
identify which rabbit residues are necessary to preserve affinity and activity. This is now
the end of the variable heavy and light chain humanized amino acid sequence.
Example 6 Inhibition of CGRP Binding to its Cellular Receptor
To characterize recombinantly expressed antibodies for their ability to inhibit
CGRP binding to its cellular receptor, a radioligand-binding assay was performed as
previously described [Elshourbagy et al, Endocrinology 139:1678 (1998); Zimmerman et
al, Peptides, 16:421 (1995)]. Membrane preparations of recombinant human CGRP
receptors, calcitonin receptor-like receptor and RAMP1 (Chemiscreen, Millipore) were
used. Antibody dilutions were preincubated with I radiolabeld human CGRPα (0.03nM)
for 30 minutes at room temperature. Non-specific binding was estimated in the presence of
0.1μM human CGRPα. Membranes were filtered and washed. The filters were then
counted to determine I radiolabeld human CGRPα specifically bound.
Results: Figure 38 demonstrates that anti-CGRP antibodies Ab1-Ab13 inhibit
CGRP binding to its cellular receptor.
Example 7 Inhibition of Neurogenic Vasodilation by Anti-CGRP Antibodies in Rats
CGRP is a potent vasodilator (Nature 313: 54-56 (1985) and Br J. Clin.
Pharmacol. 26(6):691-5. (1988)). A pharmacodynamic assay to measure CGRP receptor
antagonist activity non-invasively was used to characterize anti-CGRP antibodies. The
model relied on changes in dermal blood flow measured using a laser Doppler imaging
following the topical application of a capsaicin solution. Capsaicin activates the transient
receptor potential vanilloid type 1 receptor (TRPV-1), producing neurogenic inflammation
and vasodilatation via the local release of vasoactive mediators including CGRP and
substance P (Br. J. Pharmacol. 110: 772-776 (1993)).
On the day prior to the vasodilatation assay, animals were dosed with the test
agent or control via IP (intraperitoneal). Following dosing, the animals were shaved and
depilated in the lower back region of their dorsal side, in an area approximately 2x6cm.
The animals were then returned to their cages overnight. On the day of test, approximately
24 hours post dosing, animals were anesthetized with isoflurane gas and placed on a
temperature controlled heating pad and fitted with a nose cone for continuous delivery of
isoflurane. A laser Doppler imager was used for the observation of vasodilatation. A beam
of coherent red light generated by a 633 nm helium-neon laser was directed to the shaved
area, a rectangle (2x6 cm), and scanned at a medium resolution mode. A baseline Doppler
scan was obtained first and the location of O-ring placement predetermined by identifying
two similar low flux areas. Two rubber O-rings (~1cm in diameter) were placed in the
selected regions and a baseline scan was performed. Immediately after completion of the
scan, 1mg of capsaicin in 5 μL of an ethanol:acetone solution (1:1) was applied within each
of the two O-rings Doppler scans were repeated at 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5,
, 27.5 and 30 minutes after the application of capsaicin. Percent change from baseline
mean Flux within each of the two O-rings, was plotted as the results of vasodilatation due
to capsaicin.
In order to test recombinantly expressed antibodies for their ability to inhibit
CGRP binding to its cellular receptor, a radioligand-binding assay was performed as
previously described.
Results: Figures 39 and 40 demonstrates that anti-CGRP antibodies Ab3 and
Ab6 reduced vasodilation in this model following capsaicin administration.
Example 8 Effect of CGRP Antibody Administration on Overactive Bladder
Experiments were conducted to assess the potential efficacy of an anti-CGRP
antibody administration on bladder continence and overactive bladder. Bladder continence
is a balance between urethral closure and detrusor muscle activity, and overactive bladder
is a condition characterized by urgency, urinary incontinence, frequency and nocturia.
Some anecdotal evidence reported in the literature suggests that CGRP may be involved
with bladder continence and may correlate to and perhaps play a causal role in overactive
bladder disease pathology. Accordingly, it was hoped that the inventive anti-CGRP
antibodies, especially given their high affinity to CGRP, would potentially help prevent or
alleviate this sometimes debilitating condition. (The evidence that that CGRP may play a
role in overactive bladder includes the fact that CGRP is present in the urinary tract, DRG
and spinal cord (Wharton et al., 1986 Neurosci (3):727) Also, C-fiber afferents are critical
for carrying impulses involved in micturition to spinal cord (Yoshida et al., 2011 J
Pharmacol Sci (112):128) and these fibers are affected by CGRP. Further, it has been
reported that intravesical administration of Botox suppresses CGRP and significantly
reduces intercontraction interval in an acetic acid induced bladder pain model (Chuang et
al., 2004 J Urol (172):1529; Chuang et al., 2009 J Urol (182):786)). Moreover, it has been
recently reported that the administration of an anti-CGRP antibody purportedly reduces the
number of bladder contractions in turpentine-oil – induced overactive bladder model
(Pfizer PCT Patent Application )).
Materials and Methods
Animals:
Female Sprague-Dawley rats (247 – 299 g) (Charles River Laboratories, Saint
Germain sur l’Arbresle, France) were delivered to the laboratory at least 5 days before the
experiments in order to be acclimatized to laboratory conditions. They were housed 3 per
cage (polypropylene type E cages size: 1032 cm ) and given food (Teklad 2016 global
rodents, Harlan, 03800 Gannat, France) and water ad libitum. Sawdust (Souralit 2912 plus,
Souralit, 17080 Girona, Spain) bedding for rodent cages was changed twice weekly. The
animal room temperature (20 ± 2 °C) was maintained with a 12/12 hour alternating light-
dark cycle (light phase 7 am: 7 pm) and relative humidity maintained at 40-70%.
Laboratory equipment
Bladder catheters were connected via a T-tube to a strain gauge MX 860
Novatrans III Gold (Medex Medical SARL, Nantes-Carquefou, France) and a syringe
pump (70-2208 Model II plus, Harvard Apparatus, Les Ullis, France and Razel R-99E,
Fisher Bioblock, Illkirch, France). Intravesical pressure was recorded continuously using a
PowerLab interface (ADInstruments Pty Ltd, Castle-Hill, Australia) and Chart software
running on a PC. Data were analyzed with Microsoft Excel ® software.
Test substances
Test Anti-CGRP antibody (Ab3)
Negative control antibody (Anti-digitoxin antibody).
Chemical reagents
Physiological saline (NaCl 0.9%) (batch n° 11043411, CAS n° 7647 5) was
purchased from B-Braun via Centravet (Lapalisse, France).
Anesthetic substances
Urethane (batch n° BCBC9294, CAS n° 516) and sodium pentobarbital
(batch n° 150A1, CAS n°76- 74-4) were supplied by Sigma-Aldrich (St Quentin Fallavier,
France) and Centravet (Lapalisse, France), respectively.
Experimental groups
Two experimental groups of 10 rats were used in the experiments. Each group
was administered 10 mg/kg of either the control or the anti-CGRP antibody:
Study design
Experimental procedure
Female rats were administered test antibody or negative control antibody
intravenously at a dose of 10 mg/kg, 18 hours prior to experiments using a tail vein
injection. Fifteen (15) hours later, rats were anesthetized with urethane (1.2 g/kg,
subcutaneous (s.c.). Three (3) hours after the s.c. administration of urethane, a
polyethylene catheter (0.58 and 0.96 mm of internal and outer diameters, respectively) was
inserted into the bladder through the dome and secured with a purse-string suture. Body
temperature was maintained at 37± 2°C (TCAT-2LV controller, Physitemp,
ADInstruments Pty Ltd., Casttle Hill, Australia) throughout the experiment.
Cystometric experiment
Cystometric investigations were performed in anesthetized female rats after
surgery. Physiological saline at room temperature was continuously infused into the
bladder at a constant flow rate (2 mL/h) for a period of at least 30 min.
At the end of the cystometric experiments, animals were sacrificed by a lethal
injection (1 mL) of sodium pentobarbital (54.7 mg/mL) (CAS n°764) followed by
cervical dislocation.
Cystometric parameters
The cystometric parameters measured were:
Amplitude of micturition (AM), i.e. pressure between threshold pressure and
Maximal pressure of micturition (mmHg),
Intercontraction interval (ICI), i.e. time between two subsequent micturitions
(sec),
Micturition frequency (MF), i.e. number of micturition contractions /15 min
(peaks/15 min).
Exclusion criteria
Two rats were excluded during the experiments: One was excluded as it
presented with bladder hyperactivity during the saline intravesical infusion, and the other
because the depth of anesthesia changed during the experiment inducing modifications of
the cystometric profile.
Analysis of results
For each rat, values for AM and ICI were calculated as the mean of the last four
or five micturitions during saline infusion. Values for MF were calculated as the mean of
micturitions obtained for two intervals of 15 minutes during saline infusion.
Results are presented as mean values ± standard error of the mean (± sem).
Figures and statistical analyses were performed using GraphPad Prism (Version 4;
GraphPad Software Inc., La Jolla, CA, USA).
Statistical comparisons of values (saline infusion) in the anti-CGRP antibody
group versus the control antibody group were performed using unpaired Student t-test.
A p<0.05 was accepted for statistical significance.
Results:
As shown in , ICI was significantly greater and MF was significantly
lower in the anti-CGRP Ab-treated group (FIGS. 41A and B respectively; p<0.05, unpaired
Student t-test). No significant difference was observed for AM between groups (C,
p>0.05, unpaired Student t-test).
These results suggest that anti-CGRP antibodies may be useful in preventing or
alleviating overactive bladder, improving urinary continency and treatment of related
urinary conditions.
Example 9 Relief of neuropathic pain in rats
Damage to the peripheral nerves often leads to chronic referred pain that is
neuropathic in origin. This pain syndrome consists of sensitivity to external stimuli (e.g.,
mechanical and/or thermal) that are not normally noxious. Consequently, neuropathic pain
is refractory to traditional analgesic approaches, making it difficult to treat.
Experimentally, neuropathic pain can be modeled in animals via surgical trauma to
peripheral nerves. The Chung model is one such system where neuropathic pain is induced
by ligation of the spinal nerves of L5 and L6.
In this Example, a spinal nerve ligation was performed on male Sprague Dawley
rats. They were tested for pain sensitivity on Day 13 (allodynia confirmation) and then
again after each administration of Ab2 using the von Frey test of mechanical allodynia to
assess possible anti-allodynic activity.
Methods
Male Sprague Dawley rats (Harlan Laboratories) weighing 200-225g at arrival,
were unpacked and placed in cages. A visual health inspection was performed on each
animal to include evaluation of the coat, extremities and orifices. Each animal was also
examined for any abnormal signs in posture or movement. All animals were found to be in
good health and were placed on study.
The rats were acclimated for a minimum of two days prior to the commencement
of the experimental procedures, with the exception of randomization body weights which
were collected the day following arrival. The animals were housed individually in clear
polycarbonate conventional cages or clear polycarbonate microisolator cages with certified
irradiated contact bedding. Food and water were provided ad libitum. Environmental
controls were set to maintain temperatures of 18° to 26°C (64° to 79°F) with a relative
humidity of 30% to 70%. A 12:12 hour light:dark cycle was maintained.
Rats were tested for baseline threshold using the von Frey filaments on Days -4
or -1 of acclimation.
On Day 0, animals underwent a spinal nerve ligation procedure. All surgeries
were performed under aseptic conditions. Prior to surgeries, the rats were anesthetized.
The back region was shaved and prepared for aseptic surgery. The rats were placed in
ventral recumbence and an incision was made just left of midline at the L4 – S2 region.
The left paraspinal muscles were separated from the spinous processes (L4 – S2). The L6-
S1 facet joint were nipped and the transverse process gently trimmed to provide space to
access theL4 & L5 spinal nerve. The left L5 and L6 spinal nerves were isolated and ligated
with 6.0 silk sutures. The incision was then closed with appropriate suture material and
skin wound clips. Post-operatively, Lactated Ringer’s Solution (3.0 – 5.0 mL) was
administered via subcutaneous injection to the animals.
All animals in Groups 1 and 2 received a von Frey test on Days -4 or -1, 13, 14,
and 17. The measurement on Day 13 was taken pre-dose. The von Frey test for mechanical
allodynia assesses anti-nociceptive properties of analgesic compounds. In this test, animals
were first habituated to the testing chamber so they were calm enough for their pain
threshold to be assessed. A technician blind to the treatment groups applied light pressure
to the left hind paw of the rat using a series of graded nylon filaments (von Frey filaments)
of increasing diameter. The filaments were pressed perpendicularly against the ventral
surface of the paw until they bent. When considered painful, the rat responds by
withdrawing its paw. Threshold allodynia was determined using the Chaplan up-down
method (Chaplan et al., J Neurosci Methods, 53:55-63, 1994), which provides the precise
force for withdrawal for each rat using a psychophysical scale of testing.
Animals were allocated into two treatment groups on Day 13, based on von Frey
scores. Any animal that had a von Frey score greater than 6g was excluded from the study.
The mean von Frey scores for each group were reviewed to ensure that the mean values
and standard deviation satisfied the assumption of homogeneity. Doses were administered
by IP injection once on Day 13 (13 days after surgery) for Group 1 (Ab2) and Group 2
(negative control antibody) (11 animals in each group; Ab2 and negative control antibodies
were administered at 10 mg/kg). Group 1 received an additional IV bolus (un-
anesthetized) injection of Ab2 on Day 17 prior to behavioral testing.
Blood samples for plasma were taken on Day 17 for Group 1 and analyzed for
Ab2 titer.
Outside of the expected surgical site observations and paw dragging associated
with the Chung surgery, no abnormal observations were documented. Treatment appeared
to have no adverse effect on overall animal health nor did it disrupt the normal weight gain
expected in rats this age.
Results
All animals that underwent baseline testing prior to surgery on Day 0 had a von
Frey score of 15 (not shown) indicating normal sensitivity. On Day 13 (prior to antibody
administration), all animals had von Frey scores lower than 6g, indicating that sensitivity to
external mechanical stimuli had developed, except for two animals which were removed
from the study. Average von Frey scores at day 13 were less than 3 g (, left group
of bars). Following testing on day 13, animals were administered Ab2 or a negative
control antibody (10 mg/kg). On days 14 and 17, von Frey scores were again tested and
were higher in the Ab2-treated animals than controls (, middle group of bars and
right group of bars, respectively).
These results indicate that treatment with an anti-CGRP antibody such as Ab2
may help prevent or alleviate neuropathic pain.
Example 10 First Experiment Assessing Effect of Anti-CGRP Antibody Administration
on Analgesia (Tail Flick Model)
Three different experiments (Examples 10-12) were conducted to assess the
potential efficacy of an anti-CGRP antibody administration on analgesia or pain. In all of
these experiments a rodent tail flick (also referred to as tail withdrawal) response model
was used as the rodent tail flick response to radiant heat is a commonly used model to
detect potentially useful analgesic agents. This assay is particularly useful to discriminate
between centrally acting morphine-like analgesics (active) and non-opioid or peripherally
acting anti-inflammatory agents (inactive). This animal model and methods and materials
used therein are described below.
Materials and Methods
Animals: Male Sprague Dawley derived male rats weighing 150 ± 20 g.
Test CGRP Antibody: Ab2
Vehicle: 15 mM Histidine 250 mM Sorbitol, pH 5.5
Analgesic Compound: Morphine
Tail Flick Response Procedures: The time (seconds) required to elicit a tail
flick response induced by focused radiant heat was measured as the pain threshold in
groups of 10 Sprague Dawley derived male rats weighing 150 ± 20 g. Baseline testing for
the tail flick response was done on Day 0. The rats that have a tail flick response of 3-5
seconds were included in the study and assigned to balanced treatment groups based on
baseline tail flick responses. A 15 second cut was used to avoid tissue damage.
Development of morphine tolerance
Each of 3 groups of 10 Male Sprague Dawley rats were dosed 2 x daily via i.p.
administration with saline vehicle (2 ml/kg) in the morning and evening. One of the 3
groups was in addition administered i.p. analgesic (morphine) at a dosage of 5 mg/kg 2 x
daily for 7 consecutive days. A second of the 3 groups of rats was administered i.p. an
anti-CGRP antibody according to the invention (Ab2) at a dosage of 10 mg/kg as a single
bolus on day 0. The rats in the different groups were then each tested for tail flick response
once per day 30 min after the morning dose.
A one-way ANOVA followed by Dunnett’s t-test is applied for comparison
between the vehicle control and test-compound treated groups. P<0.05 is considered
significant.
The results of these experiments are shown in Figure 43. The results therein
indicate that the test CGRP antibody when administered at 10 mg/kg elicited significant
long-lasting analgesic effect to a thermal pain stimulus. Terminal blood samples were taken
from all the tested rats via cardiac puncture and later analyzed for Ab2 titer.
Example 11: Second Tail Flick Experiment Assessing Effect of-CGRP Antibody on
Analgesia) (Antibody Dose Titration)
A second set of tail flick experiments were conducted to assess the effects of
different anti-CGRP antibody dosages on analgesia using an anti- CGRP antibody
according to the invention (Ab2). The rats used in these experiments are the same type as
in the previous experiment and the tail flick protocol substantially the same. In this
experiment analgesia was compared in different groups of animals administered different
anti-CGRP antibody dosages in order to assess whether the dosage has an effect on
analgesia. In the second set of experiments, five groups of test animals were compared as
follows. A first control group of animals were each administered the vehicle alone (15 mM
Histidine 250 mM Sorbitol, pH 5.5), 3 groups of animals were each administered different
dosages of the same anti-CGRP antibody contained in the vehicle (Ab2, respectively
administered at dosages of 1 mg/kg, 3 mg/kg or 10 mg/kg on Day 0), and a fifth group of
animals was administered 10 mg/kg of a negative control antibody (anti-digitoxin
antibody) also on Day 0.
The tail flick protocols were otherwise substantially effected as above-described.
The results were again assessed using one-way ANOVA followed by Dunnett’s t-test for
comparison between the vehicle control, negative control antibody and test-CGRP antibody
treated groups. P<0.05 is considered significant.
The results of these experiments are shown in Figure 44. It can be seen
therefrom that the higher antibody dosages of the test compound (inventive Ab2 anti-
CGRP antibody) elicited better analgesic effects than the lower dosages. As anticipated the
negative control antibody did not elicit a perceptible effect on analgesia relative to the
control groups.
Example 12: Third Tail Flick Experiment Assessing Effect of Anti-CGRP
Antibody/Morphine Co-administration on Analgesia
A third set of tail flick experiments were also conducted to assess the effects of
anti-CGRP antibody/morphine co-administration on analgesia. In these experiments a first
group of animals was administered the same vehicle alone at a dosage of 5 ml/kg. A
second group of animals was administered morphine on days 1-10 at a dosage of 5 mg/kg,
administered twice daily, wherein such animals were on Day 0 were also administered the
anti-CGRP antibody Ab2 at a dosage of 10 mg/kg. A third group of animals was
administered morphine on only days 1-4 again at a dosage concentration of 5 mg/kg,
administered twice daily, and were further administered the Ab2 antibody on Day 0, at a
dosage of 10 mg/kg. All administrations were i.p.
Tail flick experiments were effected in each of these groups of animals daily
from Day 0-10. The results of these tail flick experiments were again assessed using one-
way ANOVA followed by Dunnett’s t-test for comparison between the vehicle control,
negative control antibody and the test anti-CGRP antibody treated group. P<0.05 is
considered significant.
The results of these comparisons are summarized in Figure 45. The Ab2-treated
animals receiving a daily dose of morphine throughout the experiment exhibited morphine
tolerance, and after day 5 the tail flick time had decreased almost to the level of vehicle-
treated control animals. In contrast, in Ab2-treated animals receiving morphine only until
day 4, the tail flick time improved on day 5 and remained improved until day 8. The
results suggest that the administration of an anti-CGRP antibody may have analgesic
effects even after onset of morphine tolerance, which may be more pronounced upon
withdrawal of morphine.
Example 13 Relief of visceral pain in rats
Patients suffering from irritable bowel syndrome (IBS) demonstrate a lower
visceral sensory threshold to colorectal balloon distension (Ritchie, Gut, 1973, 14:125-32).
It has been suggested in IBS that there is heightened pain sensitivity of the brain-gut axis,
with a normal pattern of activation. It has previously been shown that injection of
trinitrobenzene sulfonic acid (TNBS) into the proximal colon provoked chronic colonic
hypersensitivity, measured in conscious rats by a decreased pain threshold in response to
colonic distension (Diop et al., J. Pharmacol. Exp. Ther., 2002, 302:1013-22). This chronic
hypersensitivity was found in the distal non-inflamed colon and persisted for 21 days. It
mimicked certain characteristics of IBS and so it can be used as a model to experimentally
explore the pathophysiological aspects of this disorder. This assay is used to determine the
potential antihypersensitive effects of compounds for TNBS-induced colonic
hypersensitivity.
Several studies have implicated CGRP in visceral pain (Friese et al., Regul Pept
1997;70:1–7; Gschossmann et al., Neurogastroenterol Motil 2001;13:229–36; Julia and
Bueno, Am J Physiol 1997;272:G141–6; Plourde et al., Am J Physiol 1997;273:G191–6).
CGRP is the most abundant peptide of capsaicin sensitive afferent fibers of gastrointestinal
origin, accounting for up to 80% of overall peptide immunoreactivity (Clague et al.,
Neurosci Lett 1985;56:63–8; Sternini et al., Gastroenterology 1987;93:852–62).
Additionally, injection of CGRP induces colonic hypersensitivity in a TNBS model
(Delafoy et al., 2006, Gut 55:940-5), which is reversed by a CGRP antagonist peptide
(CGRP 8-37).
This example describes testing of an anti-CGRP antibody in a model of visceral
pain (TNBS-induced chronic colonic hypersensitivity) in rats.
Methods
Male Sprague-Dawley rats, weighing 390 to 450 g the day of surgery were
included in this study. They were housed in a temperature (19.5°C - 24.5°C) and relative
humidity (45 % - 65 %) controlled room with a 12 h - light/dark cycle. Animals were
housed 2 or 3 per cage and an acclimation period (at least 5 days) was observed before
testing. Each rat was identified by tail markings. The study was performed according to
the guidelines of the Committee for Research and Ethical Issue of the I.A.S.P. (1983) and
the European guidelines 2010/63/UE.
Colonic sensitivity was induced by surgical administration of Trinitrobenzene
sulfonic acid (TNBS, 50 mg/kg) 7 days before behavioral testing. Fasted (24 hours)
animals underwent surgery. Briefly, under anesthesia (Acepromazine 5 mg/kg / Ketamine
mg/kg), injection of TNBS (50 mg/kg, 1 ml/kg) was performed into the proximal part of
the colon (1 cm from the caecum). After surgery, animals were returned to their cages in a
regulated environment, and fed ad libitum until the testing day, 7 days later. “Naïve”
animals (rats without surgery) were placed in the same housing conditions.
Animals were administered the anti-CGRP antibody Ab2 or a negative control
antibody (both at 10 mg/kg) intravenously 24 hours prior to determination of colonic
threshold. Three groups of rats were included in this study:
Group 1: A “Naïve” group composed of animals that did not undergo surgery or
TNBS treatment on D-7 and were treated with the control antibody 24 hrs prior (i.e. D-1) to
testing (i.e. measurements of the colonic distention threshold on D0) (n=7).
Group 2: A “TNBS” group composed of animals that underwent surgery on D-7
and were treated with control antibody (24 hrs prior (i.e. D-1) to testing (i.e. measurements
of the colonic distention threshold on D0) (n=8).
Group 3: A “Treated” group composed of animals that underwent surgery on D-
7 and were treated with Ab2 24 hrs prior (i.e. D-1) to testing day (i.e. measurements of the
colonic distention threshold on D0) (n=8).
Seven days (D7) after TNBS injection, colonic sensitivity was assessed by
measuring the intra-colonic pressure required to induce a behavioral response during
colonic distension due to the inflation of a balloon introduced in the colon. The tests were
conducted by a blinded experimenter. This response is characterized by an elevation of the
hind part of the animal body and a clearly visible abdominal contraction corresponding to
severe contractions (Al Chaer et al., Gastroenterology 2000, 119:1276-1285) and used as a
pain marker (Bourdu et al., Gastroenterology. 2005:128, 1996-2008). The balloon (5 cm)
was inserted intrarectally in a minimally invasive manner to 10 cm from the anus of fasted
(24h) vigil animals, and the catheter was taped to the base of the tail. Rats were then placed
in the middle of a plexiglass box and the catheter was connected to an electronic barostat
apparatus. After a 30 min-acclimation period with the inserted balloon, colonic pressure
was gradually increased by 5 mmHg steps every 30 sec from 5 to 75 mmHg (cut off) until
pain behavior is evidenced. Four determinations were performed, 30 min, 50 min, 70 min
and 90 min after balloon insertion.
Using the data from each test, the percentage of activity on colonic
hypersensitivity induced by the intracolonic administration of TNBS was calculated as
follows
DistentionthresholdTreated−DistentionthresholdTNBS
(Activity percentage)Treated= x100
DistentionthresholdNaive−DistentionthresholdTNBS
Distention threshold is the arithmetic mean of the values for the “Treated”
Treated
group; Distention threshold is the arithmetic mean of the values for the “TNBS” group;
TNBS
and Distention threshold is the arithmetic mean of the values for the “Naïve” group.
Naive
Results
The ability of an anti-CGRP antibody to alleviate visceral pain was tested in a rat
model in which chronic colonic hypersensitivity was induced by administration of TNBS.
Visceral pain was quantified by measuring the colonic distension threshold, i.e., the amount
of abdominal pressure that the animals could tolerate before exhibiting a behavioral
response (muscle contraction). Higher colonic distension threshold values indicate less
sensitivity. As expected, TNBS treatment resulted in greatly decreased the colonic
distension threshold compared to naïve animals (, compare middle bar (TNBS
treated) and left bar (naïve)). Ab2 administration improved the colonic distension
threshold compared to control animals (, compare right bar (Ab2 treated) and
middle bar (control)). The improvement from Ab2 administration was statistically
significant (p < 0.05 Student’s t-test, comparison to TNBS + Negative control group). The
antihypersensitive activity of Ab2 was computed to be 27% (indicative of the degree of
relief of the TNBS-induced hypersensitivity). These results suggest that anti-CGRP
antibodies may be useful in preventing or alleviating visceral pain.
The term “comprising” as used in this specification and claims means
“consisting at least in part of”. When interpreting statements in this specification, and
claims which include the term “comprising”, it is to be understood that other features that
are additional to the features prefaced by this term in each statement or claim may also be
present. Related terms such as “comprise” and “comprised” are to be interpreted in similar
manner.
In this specification where reference has been made to patent specifications,
other external documents, or other sources of information, this is generally for the purpose
of providing a context for discussing the features of the invention. Unless specifically
stated otherwise, reference to such external documents is not to be construed as an
admission that such documents, or such sources of information, in any jurisdiction, are
prior art, or form part of the common general knowledge in the art.
In the description in this specification reference may be made to subject matter
that is not within the scope of the claims of the current application. That subject matter
should be readily identifiable by a person skilled in the art and may assist in putting into
practice the invention as defined in the claims of this application.
Claims (38)
1.) An isolated anti-human CGRP antibody or antibody fragment comprising: (a) a V chain comprising the complementarity determining region (CDR) 1 sequence of SEQ ID NO: 25, the CDR 2 sequence of SEQ ID NO: 26, and the CDR 3 sequence of SEQ ID NO: 27, except that one or two of the CDR amino acid residues in said V chain has been substituted with another amino acid residue; and (b) a V chain comprising the CDR 1 sequence of SEQ ID NO: 28, the CDR 2 sequence of SEQ ID NO: 29, and the CDR 3 sequence of SEQ ID NO: 30, except that one or two of the CDR amino acid residues in said V chain has been substituted with another amino acid residue.
2.) An isolated anti-CGRP antibody or antibody fragment comprising: (a) a V chain comprising the CDR 1 sequence of SEQ ID NO: 55, the CDR 2 sequence of SEQ ID NO: 56, and the CDR 3 sequence of SEQ ID NO: 57, except that one or two of the CDR amino acid residues in said VL chain has been substituted with another amino acid residue; and (b) a V chain comprising the CDR 1 sequence of SEQ ID NO: 58, the CDR 2 sequence of SEQ ID NO: 59, and the CDR 3 sequence of SEQ ID NO: 60, except that one or two of the CDR amino acid residues in said V chain has been substituted with another amino acid residue, wherein said anti-human CGRP antibody or antibody fragment specifically binds CGRP.
3.) The anti-human CGRP antibody or antibody fragment of claims 1 or 2, wherein only one of the amino acid residues in the CDRs of said V polypeptide sequence is modified.
4.) The anti-human CGRP antibody or antibody fragment of claims 1 or 2, wherein only one of the amino acid residues in the CDRs of said V polypeptide sequence is modified. 17 7
5.) The anti-human CGRP antibody or antibody fragment of claims 1 or 2, wherein only one of the amino acid residues in the CDRs of said V and said V polypeptide sequence polypeptide is modified.
6.) The antibody fragment of any one of claims 1 to 5, wherein said fragment is selected from a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv, a camelbody, a nanobody, or a monovalent antibody.
7.) The antibody fragment of claim 6, wherein said fragment is a Fab fragment.
8.) The antibody fragment of claim 6, wherein said fragment is an scFv.
9.) The anti-human CGRP antibody of any one of claims 1 to 5, which contains an Fc region that has been modified to alter effector function, half-life, proteolysis, and/or glycosylation.
10.) The anti-human CGRP antibody or antibody fragment of any one of claims 1 to 9, which is humanized.
11.) The anti-human CGRP antibody of claim 10, wherein said humanized antibody comprises a human Fc.
12.) The anti-human CGRP antibody of claim 11, wherein said human Fc is derived from IgG1, IgG2, IgG3, or IgG4.
13.) Use of at least one anti-human CGRP antibody or antibody fragment according to any one of claims 1 to 12, for the manufacture of a medicament to treat a disease or condition associated with cells that bind or express CGRP. 17 8
14.) Use of at least one anti-human CGRP antibody or antibody fragment according to any one of claims 1 to 12, for the manufacture of a medicament to treat a headache condition.
15.) Use of at least one anti-human CGRP antibody or antibody fragment according to any one of claims 1 to 12, for the manufacture of a medicament to treat a migraine condition.
16.) The use of claim 13, wherein the disease or condition is selected from a pain related disease or condition, overactive bladder, migraine with aura, migraine without aura, cancer or tumors, angiogenesis associated with cancer or tumor growth, angiogenesis associated with cancer or tumor survival, migraine, chronic migraine, frequent episodic migraines, menstrual migraines, hemiplegic migraines, cluster headaches, migrainous neuralgia, chronic headaches, tension headaches, general headaches, hot flushes, chronic paroxysomal hemicrania, secondary headaches due to an underlying structural problem in the head, secondary headaches due to an underlying structural problem in the neck, cranial neuralgia, sinus headaches, allergy-induced headaches, allergy-induced migraines, pain, inflammatory pain, post-operative incision pain, complex regional pain syndrome, cancer pain, primary bone cancer pain, metastatic bone cancer pain, fracture pain, osteoporotic fracture pain, pain resulting from burn, osteoporosis, gout joint pain, pain associated with sickle cell crises, hepatocellular carcinoma, breast cancer, liver cirrhosis, neurogenic pain, neuropathic pain, nociceptic pain, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, fibromyalgia, menstrual pain, ovarialgia, reflex sympathetic dystrophy, neurogenic pain, osteoarthritis pain, rheumatoid arthritis pain, lower back pain, diabetic neuropathy, sciatica, visceral pain associated with gastro-esophageal reflux, dyspepsia, irritable bowel syndrome, inflammatory bowel disease, Crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstrual period, labor, menopause, prostatitis, pancreatitis, urinary incontinence, chronic pain, inflammatory pain, eye pain, tooth pain, post-surgical pain, trauma related pain, arthritis, allergic dermatitis, psoriasis, pruritus, erythema, and dysmenorrhea. 17 9
17.) A method of making the anti-human CGRP antibody or antibody fragment of any one of claims 1 to 12 in a polyploid yeast culture that stably expresses and secretes into the culture medium at least 10-25 mg/liter of said antibody, comprising: (i) introducing at least one expression vector containing one or more heterologous polynucleotides encoding said antibody operably linked to a promoter and a signal sequence into a haploid yeast cell; (ii) producing by mating or spheroplast fusion a polyploidal yeast from said first and/or second haploid yeast cell; (iii) selecting polyploidal yeast cells that stably express said antibody; and (iv) producing stable polyploidal yeast cultures from said polyploidal yeast cells that stably express said antibody into the culture medium.
18.) The method of claim 17, wherein said yeast genera is Pichia.
19.) The method of claim 18, wherein the species of Pichia is selected from Pichia pastoris, Pichia methanolica and Hansenula polymorpha (Pichia angusta).
20.) The method of claim 18, wherein the species of Pichia is Pichia pastoris.
21.) An isolated polynucleotide comprising a polynucleotide sequence encoding an anti- human CGRP antibody or antibody fragment of any one of claims 1 to 12.
22.) A vector or isolated host cell comprising the polynucleotide sequence of claim 21.
23.) The isolated host cell of claim 22, wherein said host cell is a yeast cell belonging to the genus Pichia.
24.) A pharmaceutical composition comprising at least one anti-human CGRP antibody or antibody fragment according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier. 18 0
25.) A diagnostic composition comprising at least one anti-human CGRP antibody or antibody fragment according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.
26.) The pharmaceutical composition of claim 24, which further comprises at least one stabilizer.
27. The diagnostic composition of claim 25, which further comprises at least one stabilizer.
28.) The pharmaceutical composition of claim 24, which is lyophilized.
29.) The diagnostic composition of claim 25, which is lyophilized.
30.) A pharmaceutical composition according to any one of claims 24, 26 and 28, for use in treating a disease or condition treatable or preventable by the administration of a CGRP antagonist, wherein said disease or condition is associated with cells that bind or express CGRP.
31.) The pharmaceutical composition of claim 30, wherein the disease or condition is selected from a pain related disease or condition, overactive bladder, migraine with aura, migraine without aura, cancer or tumors, angiogenesis associated with cancer or tumor growth, angiogenesis associated with cancer or tumor survival, migraine, chronic migraine, frequent episodic migraines, menstrual migraines, hemiplegic migraines, cluster headaches, migrainous neuralgia, chronic headaches, tension headaches, general headaches, hot flushes, chronic paroxysomal hemicrania, secondary headaches due to an underlying structural problem in the head, secondary headaches due to an underlying structural problem in the neck, cranial neuralgia, sinus headaches, allergy-induced headaches, allergy-induced migraines, pain, inflammatory pain, post-operative incision pain, complex regional pain syndrome, cancer pain, primary bone cancer pain, metastatic bone cancer 18 1 pain, fracture pain, osteoporotic fracture pain, pain resulting from burn, osteoporosis, gout joint pain, pain associated with sickle cell crises, hepatocellular carcinoma, breast cancer, liver cirrhosis, neurogenic pain, neuropathic pain, nociceptic pain, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, fibromyalgia, menstrual pain, ovarialgia, reflex sympathetic dystrophy, neurogenic pain, osteoarthritis pain, rheumatoid arthritis pain, lower back pain, diabetic neuropathy, sciatica, visceral pain associated with gastro- esophageal reflux, dyspepsia, irritable bowel syndrome, inflammatory bowel disease, Crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstrual period, labor, menopause, prostatitis, pancreatitis, urinary incontinence, chronic pain, inflammatory pain, eye pain, tooth pain, post-surgical pain, trauma related pain, arthritis, allergic dermatitis, psoriasis, pruritus, erythema, and dysmenorrhea.
32.) The pharmaceutical composition of claim 30, wherein the composition is useful in a therapeutic regimen for treatment of a specific disease or condition associated with pain that includes the administration of another therapeutic agent.
33.) An isolated anti-human CGRP antibody or antibody fragment as claimed in claim 1 or claim 2 substantially as herein described or exemplified and with or withour reference to the accompanying drawings.
34.) A use as claimed in any one of claims 13-15 substantially as herein described or exemplified and with or withour reference to the accompanying drawings.
35.) A method as claimed in claim 17 substantially as herein described or exemplified and with or withour reference to the accompanying drawings.
36.) An isolated polynucleotide as claimed in claim 21 substantially as herein described or exemplified and with or withour reference to the accompanying drawings. 18 2
37.) A vector or isolated host cell as claimed in claim 22 substantially as herein described or exemplified and with or withour reference to the accompanying drawings.
38.) A composition as claimed in claim 24 or claim 25 substantially as herein described or exemplified and with or withour reference to the accompanying drawings. 18 3
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